Flow cytometry as an analytical and preparative tool in immunology

Journal o f Immunological Methods, 50 (1982) R85--R112

R85

Elsevier Biomedical Press Review article FLOW CYTOMETRY AS AN ANALYTICAL AND PREPARATIVE TOOL IN IMMUNOLOGY

MICHAEL R. LOKEN and ALAN M. STALL La Rabida-University o f Chicago Institute, and the Department o f Microbiology, University o f Chicago, Chicago, IL 60649, U.S.A.

(Received 30 December 1981, accepted 8 January 1982)

Key words: fluorescence-activated

cell sorter-- immunofluorescence -- forward ligh t scattering -- laser optics -- monoclonal antibodies

angle

INTRODUCTION The use of flow systems to study cell populations has expanded rapidly in recent years. Several comprehensive and detailed reviews have been written about flow c y t o m e t r y and its application to the study of biological problems (Herzenberg et al., 1976; Horan and Wheeless, 1977; Herzenberg and Herzenberg, 1978; Melamed et al., 1979; Laerum et al., 1980). Other sources of technical information include the journal C y t o m e t r y , which is devoted to a u t o m a t e d techniques used in cell analysis, and the Flow Systems News Letter, which compiles the abstracts of papers utilizing flow systems. Since several detailed reviews are available, this article will focus on some topics which have not been covered previously, emphasizing the practical aspects of efficiently operating a cell sorter. Standard optical configurations and techniques will be discussed so that the readers will have a basis upon which to m o d i f y and extend the instrument for their particular biological problems. The techniques and procedures which are c o m m o n l y used in flow c y t o m e t r y laboratories have evolved over several years of use. Some of these did n o t originate with the authors but have been included in this article for completeness. The discussion will be limited to systems in which cells flow orthogonally through a laser beam (Kamentsky et al., 1965; Bonner et al., 1972; Steinkamp et al., 1973). Currently, 3 commercial instruments have this configuration (Becton-Dickinson, FACS Division, Sunnyvale, CA; Coulter Electronics, Hialeah, FL; Ortho Diagnostics Systems, Westwood, MA). Two cell analyzers which utilize conventional fight sources (Gohde, 1973; Steen et al., 1980) will not be discussed, although some of the concepts presented here may be directly applicable to these systems. The use of Coulter electronic volume 0022-1759/82/0000--0000/$02.75

© 1982 Elsevier Biomedical Press

R86 sensing has been extensively discussed elsewhere (Kachel, 1979) and will not be treated. ILLUMINATION The argon ion laser and mercury arc lamp represent the 2 most widely used sources of illumination in flow c y t o m e t r y . The mercury arc lamp is less expensive but is less stable and the usable light intensity is less than can be obtained from a laser. The primary reasons for selecting a laser as the illuminating light source in flow systems are the stability of the light beam and the ability to focus this beam down to the dimensions of a cell (Heibert, 1979). The intensity difference between a laser and a mercury arc lamp becomes significant in the analysis of immunofluorescence and is less import a n t when DNA specific fluorescent stains are used. DNA stains such as propidium iodide or ethidium bromide emit amounts of light orders of magnitude greater than do immunofluorescent stains. As a result, the DNA stains can be observed using an illumination system having a lower intensity beam. A commercial instrument designed to quantify immunofluorescence using a mercury lamp has recently been introduced (Becton-Dickinson). Since lasers have been the light source of choice in flow cytometers, it is necessary to understand some basic principles of laser optics. The intensity of light as measured across a laser beam is not uniform. The light flux is greatest at the center and decreases progressively from this central point (Marshall, 1971). This intensity change approximates a Gaussian curve described by equation (1): I(r ) = Ioe-2r2/tg

(1)

where I0 is the intensity at the center of the beam; I is the intensity at a radius, r; and r0 is the radius where I = 0.135 I0 (Fig. 1). The diameter (D) of a laser beam is expressed in terms of the points where the intensity has dropped to 1/e 2 (D = 2r0). For an argon ion laser (Model 164-05, SpectraPhysics, Mountain View, CA), the diameter of the laser beam is 1.5 mm at 514.9 nm and 1.2 mm at 547.9 nm (Spectra-Physics Instruction Manual, 1976). A lens, or series of lenses, is used to focus the beam onto the path of the cell. The beam cannot be focused to an infinitely small spot as a result of diffraction limitations. This departure from classical optics is illustrated in Fig. 2. The diameter of the focused spot (do) is dependent upon the focal length of the lens (F), wavelength of illuminating light (X) and initial beam diameter (D), equation (2): 4 XF do = - - X - 7r D

(2)

The standard focusing optics for the FACS incorporates a 125 mm focal

R87

I0-

/ ii

/

,//" "~xx

\ xx

il

0.135 . . . . . Io I

25p.rn

50Fro

I

0

25~rn

5C ~rn

Fig. 1. Laser beam shape. U n d e r normal operating c o n d i t i o n s the intensity of light across a laser beam has a Gaussian shape (solid line). The diameter of the beam is expressed in terms of the distance where the intensity drops to 1/e 2 (0.135) of the central intensity. When the laser optics are misaligned the intensity distribution changes. The d o t t e d line a p p r o x i m a t e d the beam shape in the TEMpi ( d o n u t ) m o d e .

length spherical lens. Using these optics the spot size at 514.5 nm is 55 pm and at 457.9 nm is 61 pm. The a p p r o x i m a t e beam size for these optics is illustrated in Fig. 1. It is evident t ha t for all cells to pass through the same l

I

_F L....

I I

!

I

Spher,cor ~eo~

( ~

°b'~r°n

,

e

I S1reom

,~

Crossed Cylmdricol

I I

Lenses

I

Fig. 2. S c h e m a t i c diagram of the laser focusing optics. A laser beam, diameter D, enters f r o m the left and passes through a lens, focal length F, to f o r m a spot, d i a m e t e r d 0. The size of the spot is limited by diffraction and is d e p e n d e n t on D, F, and the wavelength of light, k. The spot shape at d o is a circle w h e n the lens is spherical and is an elipse w h e n 2 crossed cylindrical lenses focus the light. Beam divergence, e, limits the lowest angle for the collection o f forward light scatter. The angles for light scatter are measured assuming the cell is the center of a sphere. L o o k i n g f r o m the cell, in the direction in which the laser b e a m is traveling, is defined as 0 ° (forward angle light scattering). The direction from which the beam originates is defined as 180 ° .

R88

intensity of illuminating light they must flow through the center of the laser beam. The concentric flow of sample and sheath fluid used in flow cytometers was designed to restrict the cells to the center of the stream (Crosland-Taylor, 1953). Fig. 1 also demonstrates the importance of alignment of the center of the stream with the center of the laser beam. If the alignment is not perfect, small positional changes between identical cells could cause large differences in the signals generated by those cells. Some instruments (Steinkamp et al., 1973) utilize crossed cylindrical lenses in place of a single spherical lens in order to minimize the difference that position contributes as cells flow through the laser beam. This results in a spot shape which is ellipsoidal rather than circular with the dimensions dependent upon the focal lengths of the 2 cylindrical lenses. The long axis of the ellipse is oriented perpendicular to the stream while the short axis is parellel to the stream. While the beam shape of the laser under normal conditions is gaussian, lasers do operate in other modes which do not have a gaussian shape (Spectra-Physics Instruction Manual, 1976). The most c o m m o n is the TEM* 1 mode in which the beam looks like a donut. A cross-section of the beam in this d o n u t mode is shown in Fig. 1; the intensity of light is lower in the center than it is further away. Operation in this mode usually occurs when the plasma tube is not properly aligned. Fig. 3 compares the oscilloscope traces of signals generated from particles flowing through a gaussian-shaped beam (TEM00) with those intersecting a beam with 2 maxima (TEM$1). Frequently the operation of the laser in the d o n u t mode is observed only as a flattening of the peak of the pulse shown in Fig. 3A rather than the dual peaks observed in Fig. 3B.

A

V 0 L T S

B

I

0

I

I

I

I

I

I0 20 /~Sec

Fig. 3. Oscilloscope traces for uniform I pm diameter beads flowing through a Gaussianshaped (TEM00) laser beam, A, and a beam operating in the TEMpi mode, B. Transition between TEM00 and TEMpi laser modes can be made by realignment of the laser plasma tube.

R89 Optimal operational conditions for flow cytometers requires the laser to be in the TEM00 mode. Changing the mode from TEM0*1 to the TEM00 is straightforward. Tuning the laser for peak power by adjusting the rear optics should force the laser back into the TEM00 mode. If the laser is still operating in the d o n u t mode, the front aperture (on Spectra-Physics lasers) should be closed until the oscilloscope traces are unimodal. (Adjustments to the laser should be made only after consulting the laser instruction manual.) Because of its expense the lifetime of the plasma tube is another important consideration in operating the laser. The lifetime of a laser plasma tube is related to the cumulative time the laser operates and the a m o u n t of current used during that operation. Running the laser at the lowest possible current increases the life of the plasma tube. This requires keeping the brewster windows and laser mirrors clean. For most immunofluorescence detection using fluorescein as the dye, 200 mW is sufficient to run most samples on a FACS instrument. This requires just under 20 A on the Spectra-Physics 16405. In order to operate in the ultra violet (UV), the laser must run at 37--40 A, which in turn, decreases the life of the plasma tube. LIGHT SCATTERING Dark field illumination has been used in microscopes to recognize cells with respect to their light scattering properties rather than to their absorption or refractive index differences identified in transmission techniques. The same principles of light scattering are used in flow systems thereby providing a parameter independent of fluorescence by which to distinguish between cell populations. The advantage of light scattering is that there is no requirement for fixing or staining the cells. Differential scattering has been used in flow systems to discriminate between live and dead cells (Julius et al., 1975; Loken and Herzenberg, 1975), to identify cell size (Mullaney and Dean, 1969, 1970; Mullaney et al., 1969), to distinguish cell types (Salzman et al., 1975a, b; Loken et al., 1976) and to identify cell a s y m m e t r y and orientation (Loken et al., 1977a). An excellent review of the theoretical and experimental aspects of light scattered from regularly shaped objects has been written by Salzman et al. (1979). The a m o u n t of light scattered at a n y angle is a complex function of size, refractive index, and reflective properties of the particle (Mie, 1908; Hodkinson and Greenleaves, 1963; Kirker, 1969; Brunsting and Mullaney, 1974). The geometric relationships involved are illustrated in Fig. 2. The cell is assumed to be at the center of a sphere as it passes through the laser. A vector drawn from the cell, along the center of the path in which the laser beam is traveling, is defined as 0 °. A vector drawn from the cell in the direction from which the laser beam originates defines 180 ° . Most of the light scattered by a cell is in the forward direction centered around 0 ° where diffraction is the predominant contribution. Refractive index becomes more important further away from 0 ° while reflection off the nucleus and cyto-

R90

plasmic constituents is significant at 90 ° . The angular dependence of light scattering intensity averaged for 30 individual t h y m o c y t e s is shown in Fig. 4. This relationship between light scatter intensity and angle for cells was experimentally determined using a scanning light scatter detection system (Loken et al., 1976). It is evident that a smooth, monotonic relationship between angle and intensity of scattered light does not exist, a result of diffraction of light by the cells. As a consequence, most of the scattered light is in the first lobe, near 0 °. Particles of different sizes generate scatter patterns which differ in the width and height of this first lobe. For smaller particles the first lobe is broader and lower than that observed for larger particles. The total a m o u n t of light scattered in this first lobe has been directly related to the size of the cell (Mullaney and Dean, 1969). Using this relationship, the light scattered between 0.5 ° and 2 ° has been used as a measurement of cell diameter (Mullaney and Dean, 1970). In most light scattering detection systems, however, the light is collected over much larger angles. The lowest angle of detection is critical because the major quantity of scattered light is in the first lobe and the position of that lobe gets closer to 0 ° as the cells become larger. Empirically it has been found that the best discrimination between live and dead lymphocytes and between different cell types in mouse bone marrow is obtained when the lower angle of detection approaches 0 ° (Loken and Herzenberg, 1975). The lower limit of this angle is dependent upon the divergence of the laser beam (e, Fig. 2), since direct laser light must not enter the detector. The divergence, in turn, is dependent upon the focal length of the laser focusing lens. A compromise between spot size and light scattering resolution is therefore required. This is accomplished in the FACS optical system by using a 125

I0000

~

<~

Y~

ho0o

I00

'

'

~'

I ' 1 ' 1 ' 1

~

I

i,

i 6 io 20 so 4o 5o 60 "z~ SCATTER ANGLE DEGREES

Fig. 4. Average angular d i s t r i b u t i o n o f light s c a t t e r e d by live t h y m o c y t e s . The m o t i o n o f a cell as it passed t h r o u g h an e x p a n d e d laser b e a m was used to scan the light s c a t t e r e d as a f u n c t i o n o f angle o n t o a d e t e c t o r ( L o k e n et al., 1976). T h i r t y individual m e d i u m - s i z e d t h y m o c y t e s were a n a l y z e d and the light s c a t t e r e d at each angle was averaged. This dist r i b u t i o n was c o r r e c t e d for t h e illuminating b e a m shape b u t was n o t c o r r e c t e d for the refractive i n d e x d i f f e r e n c e at t h e stream-air interface. A b e a m s t o p e l i m i n a t e d direct laser light f r o m e n t e r i n g t h e d e t e c t o r so t h a t t h e first p e a k was n o t m a x i m u m at 0 °. The rapid d r o p in i n t e n s i t y as a f u n c t i o n o f angle illustrates w h y the l o w e r angle o f c o l l e c t i o n of f o r w a r d s c a t t e r e d light is m o r e i m p o r t a n t t h a n t h e u p p e r angle o f d e t e c t i o n .

R91 mm focal length lens. With this lens the lower angle of scatter detection is 0.7 ° . It can be seen from Fig. 4 that the outer limit of collection is not as critical as the lower limit. The intensity at 15 ° is 2 orders of magnitude less than the peak intensity. It has been demonstrated that changing the outer collection angle between 8 ° and 15 ° causes essentially no change in the scatter signal (Loken and Herzenberg, 1975). If the outer collection angle is decreased below 8 ° , small positional changes of the cell, which are amplified by the lens effect of the stream-air interface, cause aberrations in the light scattering histograms. The wavelength of the illuminating light is another critical parameter in light scattering. The object size, (a) in light scattering theory is expressed in terms of the wavelength = ~d/~

(3)

where d is the particle diameter and ~ is the wavelength of light in the medium. In addition, the refractive index of the cells is also dependent upon the wavelength. Since both particle size and refractive index are wavelength dependent, it was not surprising to find differences in light scattering histograms when changes in the wavelength of laser light were studied. This was especially apparent when UV and visible light scattering were compared (Loken and Houck, 1981). The histogram for forward angle 488 nm scattered light for mouse bone marrow is compared to the histogram for UV scattered light in Fig. 5. Only 2 distinct populations can be observed using UV scattered light, while 3 peaks are apparent in the 488 nm histogram. This indicates that less discrimination between unfixed mouse bone marrow cell populations can be made using UV light as compared with 488 nm light. The relationships between the signals generated by the 2 colors of light were determined by modifying the FACS IV optics (Loken and Houck, 1981) and are shown in Fig. 6. The cluster of dots with the smallest signals in both UV and 488 nm were erythrocytes (Fig. 6,E). Two other populations, (Fig. 6,L and G), which are easily distinguished by visible light scattering, were not separated by the UV scattering signals. The cells comprising group L were predominantly l y m p h o c y t e s while those in group G were myeloperoxidase positive (Kaplow, 1965, 1 9 7 9 ) g r a n u l o c y t e s and monocytes. It should be noted that although granulocytes are physically larger than lymphocytes, by UV light scattering t h e y appear smaller than the lymphoid cells. A third population, LL, was identified b y the correlation between UV and 488 nm light scatter which did not stain for myeloperoxidase and had predominantly lymphoid morphology. Although viable and non-viable lymphocytes are easily distinguished using visible light (Julius et al., 1975; Loken and Herzenberg, 1975), this distinction cannot be made using UV light. Interestingly, by correlating UV and visible scattered light, viable T and B l y m p h o c y t e s in the spleen and lymph node could be distinguished w i t h o u t staining the cells (Loken and Houck, 1981).

R92

I

A

c.)

n-

_J LIGHT SCATTERING INTENSITY

Light Scattering 488nm

Fig. 5. Light scattering histograms of mouse bone m a r r o w cells collected from 0.7 ° to 15 °. A: UV light scattering; B: 488 n m light scattering ( L o k e n and Houck, 1981}. Fig. 6. Correlation plot of UV vs. visible light scattering signals for viable mouse bone m a r r o w cells. Dead cells, stained with p r o p i d i u m iodide, were gated out of this plot. The cells in the clusters were isolated for m o r p h o l o g i c a l and histochemical analysis. E: erythr o c y t e s , L : l y m p h o c y t e s , LL: large l y m p h o c y t e s ; G : granulocytes and m o n o c y t e s ( L o k e n and H o u c k , 1981).

These examples illustrate the necessity of considering the wavelength at which measurements are made when interpreting light scattering histograms. A change in wavelength may place different emphasis on the diffraction and refraction c o m p o n e n t s of light scattering, so that different properties of the cells may be examined at the different wavelengths. These data show that the light scattering histograms may change when the illuminating wavelength is changed on the B-D and Coulter instruments. This difficulty is averted on the Ortho instruments since an independent helium-neon laser is used to generate the light scattering signal. FLUORESCENCE

Although the argon ion laser emits monochromatic light, the wavelength can be varied. The optimal wavelength for a specific experiment is related to the absorption spectrum of the particular dyes used to stain the cells. Table 1 lists the available wavelengths on an argon ion laser, the relative intensity of each line, and a few experimental uses and normal operating powers for those wavelengths. Four of the 9 wavelengths are c o m m o n l y used in immunological experiments. For some experimental applications it is necessary to operate in the UV, which requires a 5 W laser. Conversion from visible to UV light involves replacing the front mirror and rear prism assembly with UV mirrors. This transition, including tuning the laser, takes a b o u t 20 min for an experienced operator. (The first time we changed from visible to UV, the procedure t o o k

R93 TABLE 1 Fluorescence excitation using an argon ion laser. Wavelengths available (nm)

Relative power a

Operating

514.5 501.7 496.5 488.0

1.0 0.20 0.35 0.75

400 --200

476.5 472.7 465.8 457.9 351.1 + 363.8 d

0.35 0.15 0.10 0.175 0.060

---50 c 40

Uses

power b

(mW) Rhodamine (rhodamine + fluorescein) --Fluorescein (fluorescein + propidium iodide) ---Mithramycin Hoechst 33342 (Hoechst 33342 + propidium iodide)

a Approximate power obtained from each wavelength using the same current relative to the power obtained at 514.5 rim. b Power output standardly used in immunological experiments. c The light stabilizing circuit on the Spectra-Physics model 164 lasers requires the light detecting diode switch to be in the UV mode when the 457.9 nm line is used. d Operation in the UV requires replacement of the front mirror and rear prism with UV optics.

a d a y a n d a half.) It is advisable t o have a laser c o m p a n y r e p r e s e n t a t i v e guide t h e e x p e r i m e n t e r t h r o u g h t h e first c h a n g e f r o m visible t o UV. In a d d i t i o n t o t h e usual single w a v e l e n g t h o p e r a t i n g m o d e , t h e laser can also o p e r a t e in an all-lines m o d e . B y replacing t h e rear prism w i t h a mirror, t h e laser o u t p u t s all t h e lines f r o m 4 5 7 . 9 n m t o 5 1 4 . 9 n m . Special mirrors have been designed t o o b t a i n all laser lines f r o m 351.1 n m to 4 8 8 n m ( L o k e n , 1 9 8 0 a ) . These mirrors have been used e x p e r i m e n t a l l y f o r determ i n i n g t h e cell c y c l e d e p e n d e n c e o f antigen e x p r e s s i o n ( L o k e n , 1 9 8 0 a ) , f o r assessing cell c y c l e d e p e n d e n c e o f sensitivity t o a n t i b o d y a n d c o m p l e m e n t ( L e i b s o n et al., 1 9 8 0 ) , a n d f o r i d e n t i f y i n g t h e w a v e l e n g t h d e p e n d e n c e o f light scattering ( L o k e n a n d H o u c k , 1 9 8 1 ) . J u s t as t h e selection o f t h e p r o p e r exciting w a v e l e n g t h is i m p o r t a n t f o r t h e o p t i m a l utilization o f f l o w c y t o m e t e r s , care m u s t be t a k e n in selecting emission filters. In o r d e r t o c h o o s e t h e p r o p e r filter, 3 q u e s t i o n s m u s t be answered ( L o k e n , 1 9 8 0 b ) . First, w h i c h filter is m o s t efficient in t r a n s m i t t i n g light f r o m t h a t f l u o r e s c e n t d y e ? S e c o n d , h o w well d o t h e filters eliminate t h e laser light s c a t t e r e d f r o m t h e cells? Third, d o t h e filters themselves f l u o r e s c e ? A simple y e t effective m e t h o d o f testing o p t i c a l filters u n d e r n o r m a l o p e r a t i n g c o n d i t i o n s o n a f l o w c y t o m e t e r is o u t l i n e d in t h e f o l l o w i n g example. D e t e r m i n i n g relative filter t r a n s m i s s i o n e f f i c i e n c y can be d o n e b y staining

R94 a population of cells or particles with the dye and empirically identifying which filters give the m a x i m u m signal. For example, histograms from fluorescein-conjugated microspheres observed through either of 2 filter sets, A or B, are compared in Fig. 7. It is evident that filter B was almost 2 times more efficient than filter A in transmitting light from the fluoresceincoupled test particles. A third filter, C, yielded a histogram very similar to curve B, Fig. 7 (data n o t shown). Since filter B and filter C were similar in transmitting light emitted from fluorescein t h e y were tested for their ability to eliminate the exciting laser light. Fixed chicken red blood cells (CRBC) provide a perfect model system to evaluate the reduction of the exciting light, since in certain orientations t h e y can reflect incident light directly into the fluorescence detector (Loken et al., 1977a). When fixed with OsO4 (15 min, 0°C with 0.5% OsO4) these cells do not fluoresce (Loken and Herzenberg, 1975), therefore, the only light detected in the fluorescence channel must come from light scattered by these cells. If scattered light passes the emission filter a characteristic correlation plot between the light scattering and the fluorescence signal is obtained (Loken et al., 1977a). As demonstrated in Fig. 8 (population I), emission filter B effectively eliminated the 488 nm light used to excite the fluorescein, i.e., no signal was observed in the fluorescence channel when the OsO4-fixed CRBC were analyzed. This dot plot is similar to the plot obtained when the light path into the detector was blocked or when filter A was used (data not shown). Filter C, however, allowed scattered light to pass, as evidenced by the large signals obtained in the fluorescence channel when the OsO4-fixed CRBC were analyzed (Fig. 8, population II). The OD4s8 of filter C was 3, hence it can be estimated that an optical filter must have an OD of approximately 5 at the exciting wavelength to effectively eliminate the light scattered into the fluorescence detector under conditions used to detect cell surface immunofluorescence. Finally, filter B was found to fluoresce when illuminated with 488 nm light. By moving the optical filter adjacent to the photo cathode (secondary filter 1, Fig. 9) the correlation plot shown in Fig. 8, population IV, was obtained. The shape of this dot plot indicates that signals detected in the fluorescence channel were related to the a m o u n t of light scattered from the CRBC. However, fluorescence is anisotropic and in this case not focused onto the detector. Simply by placing the filter in the primary filter holder (Fig. 9) it was possible to reduce the fluorescence originating from the filter (population III, Fig. 8). In summary, filter B is the filter of choice for use with fluorescein, however it is necessary to position it far enough from the detector so t h a t the fluorescence coming from the filter itself is minimized. New filters should always be tested for efficiency in the transmission of the fluorescent light and the elimination of scattered light. Filter fluorescence must also be ascertained to make certain it does not affect the experimental results. Proper filter positioning and filter orientation (some interference filters have a front and back

R95

~b

o

A z ~

1

o Ob n,"

.

Fluorescence Intensity

~11T

>

Light Scattering Signal

Fig. 7. Fluorescence histograms of 104 fluorescein-conjugated 1 p m spheres. The emission filters for curve A were 520 LP and 520 series ' D ' (Ditric); while for curve B OG 515 (Schott) was used. The multiple peaks observed in the curves result from detection of either singlets, doublets, or triplets. The signal from the test particles could almost be doubled solely by changing the emission filters (Loken, 1980b). Fig. 8. Correlation plots between the light scatter and fluorescence signals for non-fluorescent, OsO4-fixed CRBC. Population I was obtained using filter B and produced no signals in the fluorescence detector. Population II was obtained using filter C as an emission filter. The large signals in the fluorescence channel result from the exciting light being reflected from the flat surface of the CRBC and passing through the emission filter. For population IV filter B was placed in the rear filter holder of the FACS detector, adjacent to the photo tube. The large signals in the fluorescence channel presumably come from fluorescence emitted by the filter. By placing filter B 6 cm away from the photo tube, in the primary filter holder, these signals were eliminated (population III). (Although populations I and II, III and IV were obtained sequentially, they were superimposed here for illustrative purposes, since they did not overlap.) (Loken, 1980b.)

s u r f a c e ) m u s t a l s o b e d e t e r m i n e d f o r e a c h f i l t e r t o r e d u c e n o i s e in t h e fluorescence channel. S o m e c o m m o n l y u s e d f i l t e r c o m b i n a t i o n s a r e l i s t e d in T a b l e 2. O t h e r optical filters or filter combinations may be more efficient than those listed, however, the listed filters were adequate for the uses described. A compens a t i o n n e t w o r k ( L o k e n e t al., 1 9 7 7 b ) is r e q u i r e d t o c o r r e c t f o r s p e c t r a l o v e r lap when fluorescein and rhodamine or Hoechst 33342 and fluorescein are u s e d as d y e p a i r s . A d i s c u s s i o n o f a d u a l l a s e r e x c i t a t i o n s y s t e m w h i c h separates the signals from the 2 dyes both by spectra and by time (Stohr, 1 9 7 6 ) is b e y o n d t h e s c o p e o f t h i s a r t i c l e . With the availability of real time logarithmic amplifiers, the usable ranges of fluorescence can be greatly extended, thereby permitting the comparison

R96

Photo- detector

I

Secondary Filter.\ #2 ~, .................

detector l

I I

LI II l

!i

i

I

il

I<

Secondary Filter

I

Beam Splitte~

i

......... :3... il

iI

: Primary Filter Fluorescence

Focusing Lens

Laser

Stream

Fig. 9. Schematic diagram of fluorescence detection optics. The specific filters used for various dye combinations are presented in Table 2.

of cell populations with widely differing fluorescence. The heterogeneity of cell surface expression of Ig on B l y m p h o c y t e s in the rabbit provides an excellent example. When spleen cells from rabbits are labeled with fluorescein conjugated goat anti-rabbit lg, a broad distribution of immunoglobulin positive cells is observed (Fig. 10). Many cells in this linear plot are off-scale because they stain t o o brightly at the gain settings required to see the dimly fluorescent cells. By analyzing this cell population using a logarithmic amplifier, all cells are represented and 2 distinct populations of stained cells are evident (Fig. 11, d o t t e d line). Interestingly the rabbit lymph node lacks the brighter staining Ig positive cells (Fig. 11, solid line). The logarithmic amplifier has also aided in demonstrating the specificity of several IgM monoclonal antibodies. Murine spleen cells stained with fluorescein conjugated polyvalent rabbit anti-mouse Ig exhibited a clear bimodal histogram (Fig. 12), the Ig positive B cells exhibit 10 U fluorescence while the Ig negative T cells express 0.1 U fluorescence. By staining the cells first with anti-Thy 1.2 (IgM) followed b y the same anti-Ig, all the cells became fluorescent and the population centered around 0.1 U was lost. In contrast, only a portion of the Ig negative T cells were labeled with anti-Lyt2 followed b y the FITC anti-Ig. The remaining Lyt-2 negative, Ig negative T cells, were clearly distinguished from the Ig positive and Lyt-2 positive cells. Thus, monoclonal antibodies which react with all T cells or a subset of T cells can be readily identified using this technique.

351 363 351 363 All lines e 351--488

H o e c h s t 33342 50% silvered m i r r o r 50% silvered m i r r o r

K V 389 c K V 389 c

None

None

580 dichroic m i r r o r d

None

10% silvered m i r r o r

10% silvered m i r r o r

None

Beam s p l i t t e r

420 SP b 440 SP b

None

None

None

KV 550 c

520 LP 530 Series ' D ' None

None

None

S e c o n d a r y filter no. 1

520 LP b 560 LP b

600 LP b

None

None

550 SP b

None

Neutral d e n s i t y as n e e d e d

600 LP b

None

S e c o n d a r y filter no. 2

a Filter p o s i t i o n s refer t o Fig. 9. b Ditric Optical Co., Marlboro, MA. c S c h o t t Optical Co., Durea, PA. d This dichroic m i r r o r has 50% T at 580 n m w h e n tilted 45 ° t o the i n c i d e n t light. S o m e 620 LP i n t e r f e r e n c e filters f r o m Ditric have these spectral characteristics. e These special laser mirrors can be o b t a i n e d f r o m B e c t o n - D i c k i n s o n Co., Sunnyvale, CA.

H o e c h s t 33342 + p r o p i d i u m iodide H o e c h s t 33342 + fluorescein

457.9

Mithramycin

LP b 530 c LP b Series ' D ' b 389 c

520 OG 520 530 KV

None

514

488

Fluorescein + 90 ° light s c a t t e r

520 LP b 530 Series ' D ' b

K V 550 c

488

Fluorescein + p r o p i d i u m iodide

520 LP b 530 Series ' D ' b

514

488

Fluorescein

P r i m a r y filter a

Rhodamine Fluorescein + rhodamine

Fluorescence excitation (nm)

Use

Optical filter c o m b i n a t i o n s used f o r various dyes.

TABLE 2

cD

R98

d~

,?

.Q

E

o

e,)

nr" 0

0:,

5 I0 FLUORESCENCE INTENSITY (Arbitrary Unils)

I00

Fluorescence Intensity

Fig. 10. Linear display of immunofluorescence histogram of rabbit spleen cells stained with fluorescein conjugated goat anti-rabbit Ig. Comparisons of the brightly stained, dimly stained and negative cell populations are difficult to make on this linear representation. Fig. 11. Logarithmic display of immunofluorescence of rabbit lymphocytes labeled with fluorescein conjugated goat anti-rabbit Ig. Dotted line: spleen; solid line: lymph node. The brightly stained population of B cells in the spleen is absent in the lymph node.

Combining light scatter with fluorescence from individual cells provides a method of identifying the reactivity of some antibodies with different cell t y p e s . T h e p r e s e n t a t i o n o f 2 p a r a m e t e r d a t a , so t h a t i t is b o t h e a s y t o u n d e r s t a n d y e t p r o v i d e s s u f f i c i e n t i n f o r m a t i o n t o r e a c h p r o p e r c o n c l u s i o n s , is

IG POSITIVE

J

U

IG ~ G A T I V E

C

::<-- AHTI ~

1,2

ANTI L~tT 2 - - - ~ + ANTI IG i

60e

E L

\ •I1

11 1o RELATIVE F-LUO~EW~ IMT~ITY

Fig. 12. Logarithmic display of immunofluorescence from mouse spleen cells. When splenocytes are labeled with tanti-total Ig, 2 populations are clearly identified. The negative population becomes positive when the splenocytes are first reacted with an IgM monoclonal anti Thy 1.2 (AT-83a, F.W. Fitch) followed by fanti-total Ig. It is evident that all the Ig negative cells bind this antibody. Only a portion of the Ig negative cells are pushed into the Ig positive population when the spleen cells are reacted with an IgM monoclonal anti-Lyt-2 (BS-3-155, F.W. Fitch) followed by fanti-total Ig. The remaining Ig negative cells have the phenotype Ig-, Lyt-2-, Thy 1.2 ÷.

R99

Z

UII hi ¢r" O I h t~ .M

Z

Forward

Angle Light Scattering

J

r~z'~

64

i,i

co 32 br--

52

64

FORWARD A N G L E LIGHT SCATTERING

Fig. 13. Comparison o f displays o f 2 parameter data. Mouse bone marrow was labeled with a rat anti-mouse m o n o c l o n a l a n t i b o d y ( D N L 3 . 7 , Loken et al., 1982) f o l l o w e d by fmouse anti-rat Ig. The correlated dot plot between forward angle light scattering and fluorescence of this data is s h o w n in A. The 3-dimensional isometric display is s h o w n in B. The contour plot o f the same data is illustrated in C. The contour lines were drawn at 2%, 5% and 10% of peak counts.

R100

sometimes difficult. Three different representations of the same data are presented in Fig. 13. The correlation, or d o t plot, is effective at showing relationships between 2 parameters yet it does n o t provide information as to the relative number of events in any cluster. The 3
Staining cells in 96-well microtiter plates, in contrast to earlier techniques requiring staining in individual test tubes, has permitted expanded experimental protocols and rapid screening of multiple samples. Using individual tubes for staining, a standard immunofluorescence experiment was usually limited to approximately 30 samples. By staining cells in a V-bottom microtiter tray, the standard immunofluorescence experiment which one person can easily handle increases b y an order of magnitude. The ability to physically handle large numbers of samples has enabled us to utilize the flow c y t o m e t e r as the primary screening assay for monoclonal antibodies directed against cell surface determinants (see the following section). The staining protocol follows the same general procedure as previously described (Loken et al., 1979}. Viable (1--2 × 106) cells are aliquoted into a flexible V-bottom microtiter tray (Dynatec, Alexandria, VA) and pelleted at 250 × g for 5 min. (Carriers for microtiter plates are available for most centrifuges.) The supernatants from each sample are removed by suction with a slight tilting of the tray. An appropriate dilution of each stain in buffer containing 0.1% NaNa and 1% BSA (w/v) is added to each well (10-25 X) and an 8-channel multiple micropipette (0.05 ml, Lab Systems, Helsinki) is used to resuspend each row. The pipette tips can be rinsed in buffer and therefore do n o t have to be changed between samples. After 15 min at 0°C 3 drops of buffer from a Pasteur pipette (approximately 100 X} are added to each well. Approximately 25 X of 10% (w/v) BSA is dropped in the center of each well. The cells are again pelleted and supernatants removed. A second step antibody can then be added and the procedure repeated. (Resuspension using a multichannel pipette appears more complete with these small volumes than using a mechanical mixer.) The cells are finally pelleted through 10% BSA and the supernatants removed. The stained cells are then transferred using a 4-channel multipipette (0.2 ml, Lab Sys-

R101

tems) to test tubes containing 0.5 ml buffer for introduction into the flow cytometer. The final resuspension buffer contains 0.1% NAN3, 1% BSA and 0.2 pm/ml propidium iodide (PI). Propidium iodide, a DNA specific stain, can only enter cells in which the plasma membrane has been damaged, therefore, non-viable cells are brightly labeled while viable cells remain unstained (Krishan, 1975; Horan and Kappler, 1977). An example of this technique is shown in Fig. 14. The stained, non-viable, cells are easily distinguished from the erythrocytes and viable lymphocytes. The flow c y t o m e t e r is then set to collect immunofluorescence of only PI negative cells. This technique is preferable to performing live/dead discrimination using forward angle light scattering. The detection of viability using forward angle light scattering can only be done for small l y m p h o c y t e s using visible light. The light scattering histograms of viable and non-viable cells overlap when large cells are analyzed; when mixtures of cell types, such as in bone marrow, are studied; and when UV light is used to illuminate the cells. Because the excitation spectrum of PI is so broad, this technique can be used with 488 nm and 514.5 nm excitation as well as with UV excitation. The discrimination between live/dead using PI does not require a 2-color detection system when used with immunofluorescence. The fluorescence from PI stained cells is so bright that at linear gain settings used to identify immunofluorescence, the dead cells are all off-scale. Therefore, the immunofluorescence from non-viable cells is not observed. Similarly, using logarithmic amplification, the dead cells have a unique very bright fluorescence which is easily distinguished from immunofluorescence on viable cells. Flow cytometric analysis of cells stained by immunofluorescence may provide clues to the introduction of artifacts in the staining procedure. One t y p e of artifact which has been observed is the formation of doublets of cells. This is detected by the presence of a second population of stained cells

er

Fig. 14. Isometric display of propidium iodide stained spleen cells. Dead cells (D), erythrocytes (E) and lymphocytes (L) can be distinguished using light scattering and PI fluorescence.

R102 64

A

T H Y 1. Z F L U~ 0 R E S C E n C E

32 64

64

B

T H Y 1. 2 F L U~ 0 R E S C E N C E

FOI~I,IqRD ~

32 Llg-Fr SCAI~II~IG

64

Fig. 15. I d e n t i f i c a t i o n o f d o u b l e t s . Murine t h y m o c y t e s were labeled with anti-Thy 1.2 f o l l o w e d b y fanti-Ig. In the c o n t o u r plot b e t w e e n light scatter and i m m u n o f l u o r e s c e n c e , the d o u b l e t s appear as a s e c o n d peak w i t h t w i c e the f l u o r e s c e n c e o f the primary peak (A). These cells can be deaggregated b y passing the cells through a 27-gauge needle (B). C o n t o u r lines were drawn at 5%, 10% and 20% o f the peak value in A, and at corresponding cell c o u n t s in B. ( A total o f 5 0 , 0 0 0 viable cells were e x a m i n e d in these displays.)

R103

N d M B

>

LY~IDHOID CELLS

ERYTT~'~ TES

o

~LOID CELLS

;

_

C E L L

S

..,/..~"".''", ~ ~ '

FOR~D Ah~LE LI(g~T SCATTER

Fig. 16. Identification of loss of cell populations. The forward angle light scattering histogram between unstained (solid line) and DNL3.7 (Loken et al., 1982) labeled mouse bone marrow cells (dotted line) indicate that many erythrocytes and a minor population of large cells is lost during the staining process.

with increased light scattering and with fluorescence 2-fold brighter than the primary peak (Fig. 15A). T he doublets can be dissociated by passing the sample th r o u g h a 27-gauge needle (Fig. 15B). This procedure does n o t cause a detectable increase in cell death as assessed by PI staining. A second t y p e of artifact which has been observed is t he agglutination o f a specific p o p u latio n o f cells in a m i xt ur e by the reactive antibodies. The loss o f a population of cells can be identified by changes in the light scattering profiles o f stained and unstained cell populations. Fig. 16 compares the light scattering histogram of unstained mouse bone marrow cells (solid line) t o those labeled with a m o n o c l o n a l a n t i b o d y which identifies an antigen expressed in high densities on e r y t h r o c y t e s ( d o t t e d line). It is evident t hat there has been a dramatic loss of the e r y t h r o c y t e s with the addition of the m o n o c l o n a l a n t i b o d y . Aggregates o f e r y t h r o c y t e s could be observed in t he staining well even with a single step stain. Further, these aggregates could n o t be dissociated by passage through the 27-gauge needle. This loss of cells could only be eliminated by using lower titers of t he m o n o c l o n a l ant i body. RAPID ANALYSIS ANTIGENS

OF

MONOCLONAL ANTIBODIES TO

CELL SURFACE

In recent years t he identification o f unique cell surface antigens (CSA) has been used b o t h as a means of studying differentiation and of identifying functionally distinct subpopulations of l y m p h o c y t e s . T he i n t r o d u c t i o n of h y b r i d o m a t e c h n o l o g y (K6hler and Milstein, 1975) has overcom e m a n y o f the problems a t t e n d a n t in preparing 'monospecific' antisera to m i nor a n d / o r

R104 immunogenically weak antigens. In screening for monoclonal antibodies (MAbs) to potentially rare CSA the assay system must be technically simple yet sensitive. MAbs are generated with such ease that it is important to rapidly identify and focus attention on those of greatest interest. The standard assays for screening have been enzyme-linked immunosorbent assay (ELISA), cellular radioimmune assay (CRIA) and cytotoxicity as measured by S~Cr release (CR) (Oi and Herzenberg, 1980). By staining cells in microtiter plates it has become practical to routinely screen for MAb to CSA by flow c y t o m e t r y since 600--1000 samples can be screened in a day. The mechanics and time involved in staining the cells for fluorescence analysis is comparable to that required for ELISA, CRIA or CR. The quantitative fluorescence analysis can be performed at rates of 200--300 samples/h. The principle advantage of screening MAbs by flow c y t o m e t r y is that samples are simultaneously analyzed for multiple cellular parameters; fluorescence (amount of MAb bound), light scatter (cell size), and viability. Because individual cells rather than bulk populations are assayed, MAbs which bind to subpopulations are readily identified. In this manner, it is possible to distinguish a MAb which binds in high concentration to a subpopulation of cells from a MAb which binds in small quantities to all the cells in a sample. In addition, by correlating the fluorescence with the light scatter signals, MAbs that bind to subpopulations which differ in cell size can be distinguished. Since the background non-specific fluorescence is distributed among cells of all sizes, the signal to noise ratio for any given subpopulation of cells is increased. Backgrounds are decreased further by using propidium iodide to prevent the analysis of dead cells in a sample. MAbs bind to dead cells at high levels and thus contribute to backgrounds in binding assays such as CRIA or ELISA. As a result of the low background and discrimination of cells b y size, it is possible b y flow c y t o m e t r y to identify MAbs which bind as few as 5--10% of the cells in a given sample. Fig. 17 outlines a strategy that has been used to rapidly screen and identify MAbs to murine leUkocyte cell surface alloantigens. In the example shown, the hybridomas were generated from a BALB/c (H-2d; IgH-Ca) mouse immunized with C57BL (H-2b; IgH-C b) spleen cells and fused with SP2/0 cells b y the m e t h o d of McKearn et al. (1979) (AF6; Table 3). The primary objective of this fusion was to produce MAbs to aUotypic determinants of cell surface immunoglobulins, however, MAbs to all alloantigenic differences between BALB/c and C57BL mice were potentially generated. Elimination of unwanted MAbs was accomplished by cell sorter analysis rather than b y the immunization procedure. Thus, MAbs to unexpected b u t relevant specificities were not unknowingly eliminated by restricting the strains used for immunization or for screening. From each well containing a macroscopic hybridoma colony, 150 #1 of supernatant was withdrawn and used for all subsequent tests. Up to 10 analyses can be run from this initial aliquot. The target cells for the primary

R105 Initial Screening on C57BI/10

spleen

I ~-'~alneg/Test~ P : : : i i v e : : m p l ~ .

I

,n e. I

*

l

I

AIIoantigen

Potential

la antigen

I

Potential

Potential

IgM

Potential IgD

I

I

I

H-2 ant gen

I Test on H-2 congenJc strains

Test on allotype type strains

Fig. 17. Strategy for the rapid screening and characterization of MAb produced from a BALB/c anti-C57BL spleen cell (AF6) fusion. See text for explanation. screening were C57BL/10 (B10) spleen cells. (In general, spleen cell preparations have a higher non,specific binding of antibodies than either lymph node cells, thymus, or bone marrow. Thus, lymphoid cells from tissues other than spleen are preferred when possible.) Rabbit anti-mouse IgG adsorbed to remove # and light chain reactivities was used as the fluorescein labeled second step antibody. The threshold (intensity of fluorescence) above which a given sample was considered to be positive for a given Ab was first established. For this purpose, positive and negative controls for the second step Ab m u s t be run on every t y p e of cell whether from different tissues or different strains of mice. In the initial screening on spleen cells, the staining profiles observed on fluorescence/light scattering d o t plots fell into 3 major categories (Fig. 18): (1) those samples which show no staining above the threshold (Fig. 18A); (2) samples in which approximately 60% of cells or less were stained (Fig. 18B). MAbs to Ia, Ig and B cell alloantigens have all been found among samples of this category; (3) samples in which more than 80% of the cells were stained (Fig. 18C). MAbs to H-2 antigens as well as other non-H-2 linked alloantigens (Fig. 19D) have been found in this group. On occasion a well contained more than one secreting hybridoma. In the d o t plot in Fig. 18D all the cells were stained, however, the fluorescence intensity of approximately 50% of the cells was significantly greater. Upon subsequent cloning of this well, MAbs to b o t h an H-2 antigen and IgD were identified.

BALB/c anti-C57BL

SJL a n t i - B A L B / c

AF6

AMS

453/562 (81%)

747/828 (90%)

T o t a l wells p r e p a r e d

Wells pos. for g r o w t h

333

600

Number of wells s c r e e n e d

0/2 a

10/10 a 54

56

H-2 linked

2/2

0/10

Non-H-2

> 80% staining

4/33 b

9/27 c

Ig

28/33

33

16/27

27

H-2 linked

< 60% staining

1/33

2/27

Other

a Of the samples s t a i n i n g > 80% only 10 f r o m t h e A F 6 fusion a n d 2 f r o m t h e AMS fusion were r a n d o m l y selected for f u r t h e r analysis. b I d e n t i f i e d by the inability t o stain cells s t r i p p e d of surface Ig. c Identified b y positive staining o n CB-17 ( B A L B / c IgH-Cb).

Immunization

Fusion

C h a r a c t e r i z a t i o n o f m o n o c l o n a l a n t i b o d i e s to cell surface antigens.

TABLE 3

R107 A.

B.

C.

D.

Z Ld

0

Z IJ.I, 0 Ld

0 --I LI.

) FORWARD A N G L E L I G H T

) SCATTER

Fig. 18. Identification of m o n o c l o n a l antibodies by f l o w c y t o m e t r y . All data was acquired at the same linear gain setting. The d o t t e d w h i t e line s h o w s the p o i n t above w h i c h cells were considered to be positively stained. A: negative control - - 1st step B S A ; B: a MAb staining a p p r o x i m a t e l y 60% of the cells; C: a M A b staining a p p r o x i m a t e l y 90% o f the cells; D: a sample in w h i c h t w o M A b ' s were present, one staining a p p r o x i m a t e l y 50% and another a p p r o x i m a t e l y 90% o f the cells.

The cells from wells meeting the initial selection criteria were transferred to 1 ml culture in 24-weU plates. Typically 10--20% of the wells analysed from a given fusion contained MAbs reactive with CSAs (Table 3). The following day tests were carried on these selected wells to further characterize the MAbs and determine which of the cell lines were to be cloned for continued study. A major problem in obtaining stable MAb-producing lines is that the MAb-producing cells are often overgrown by non-secreting hybridomas in the Same well. Therefore, it is important for the secreting cell lines to be cloned as soon as possible. With such a high percentage of the wells positive for MAb activity, it was not practical to clone all the wells positive for MAb activity, thus, it was important to rapidly characterize and identify those wells of greatest interest. Once the cells were cloned and frozen, they could be further characterized. To determine if MAbs recognized an antigen linked to the major histocompatibility complex (MHC) (i.e., H-2 or Ia antigens) samples were tested on B10.D2 spleen cells. B10.D2 mice are H-2 congenic with C57BL and express the H-2 d haplotype which is identical to BALB/c (Klein, 1975). MAbs derived from this fusion which were of BALB/c (H-2 d) origin should not bind to cells expressing that same H-2 haplotype if they are reactive with

R108

r L U E $ ,2

0 --N S

I

I

I

I

I

I

I

T

I

I

'

i

,

Z~

°

o

•

64

1

:~2

F L U 0 R E C E r4

C E~

T tN S

1 Y

FOI~,I~ AI61JE L~161-rfSC.qlrn~

FC6~I~) ANGLE LIGHT SCATTER

Fig. 19. Monoclonal antibodies to mouse cell surface alloantigens. A: AMS 32.1, anti I-Adj'; B: AF3-44.1; C: AF4-62.4, anti-H-2Dd'U; D: AMS 22.1. Analyses were carried out on BALB/c (A, C, and D) or A/J (B) bone marrow cells.

MHC-linked antigens. Therefore, MAbs which stained B10 but not B10.D2 spleen cells were considered to be reactive with MHC antigens. Those that stained approximately 50% of the cells were probably anti-Ia antigen (Hammerling, 1974) while those staining >80% of the cells were probably anti-H-2 antigen. In a similar manner, linkage to the Igh-C locus was determined using allotype congenic mice. C.B-17 mice are congenic with BALB/c except for the IgH-C b locus of C57BL. Thus, a MAb from BALB/c anti C57BL fusion could only react with C.B-17 cells if it recognized an allotype-linked marker, presumably IgM or IgD. An alternative method for identifying anti-aUotype MAbs was to compare the staining of normal spleen cells to the staining of

R109

spleen cells stripped of their surface Ig b y incubation with anti-Ig for 30 min at 37°C (Taylor et al., 1971). MAbs directed against surface Ig b o u n d to the normal cells b u t n o t to the cells stripped of their surface Ig. (It was important to run controls to be assured that all of the surface Ig has been stripped.) No discrepancy in the identification of MAbs to surface Ig by the 2 methods has been observed. The ability of normal mouse serum to c o m p e t e for binding was used to distinguish between probable anti-IgD allotypes and anti-IgM allotypes. Normal serum contains almost no IgD and cannot compete o u t staining b y MAbs anti-IgD allotypes, however, staining by Abs to surface IgM allotypes is totally blocked by serum IgM (Herzenberg et al., 1977). Therefore, if binding b y a MAb to an allotype-linked antigen is blocked b y normal sera, it is initially presumed to be anti-IgM. With these few tests, which were carried o u t in 2 days using supernatant from the original well, a rapid initial characterization of the MAbs was accomplished. MAbs to Ia, H-2 and Ig were identified and could be saved or eliminated from further study depending u p o n the interests of the investigator. Only those wells of interest were cloned. Once stable MAb-producing lines were obtained t h e y were further tested on either a series of recombinant H-2 congenic strains to map the antigen (i.e., I-A, I-E, H-2K, H-2D) recognized, or allotype t y p e strains (Green, 1979) to identify the allotypic specificity recognized. Monoclonal antibodies to alloantigens found on bone marrow cells have also been produced. As with spleen cells, when bone marrow cells were used as target cells in the initial screening, certain staining patterns were commonly observed. The reactivities fell into 5 major categories (see Fig. 19A-D). A: this staining profile is typical o f MAbs to Ia antigens or surface IgM. The MAb binds to approximately 15% of the nucleated cells (dashed box). B: AF3-44.1-a MAb from a BALB/c anti-A/J fusion binds to approximately 80% of the large nucleated cells. Preliminary studies indicate that it recognizes an alloantigen found primarily on cells of myeloid origin. C: this pattern is typical of MAbs recognizing H-2 antigens. MAbs to non-H-2 linked determinants showing this pattern have also been identified. D: AMS 22.1. This MAb recognizes an alloantigen found on all spleen cells, however, in bone marrow 5 different subpopulations of nucleated cells can be identified. Work is in progress to correlate the subpopulations identified b y this MAb and AF3-44.1 with other MAbs to hemopoietic cell subsets generated in this lab (Loken et al., 1982). MAbs to some B cell antigens have yet another pattern, staining approximately 70% of the small lymphoid cells in the bone marrow (data not shown) (Dessner and Loken, 1981). A summary of the results of 2 fusions is presented in Table 3. The MAbs were characterized following the strategy outlined. As might be expected, more than 95% of the MAbs identified recognized Ig, Ia or H-2 antigens. The other 5% recognized other genetic polymorphisms. Differences from fusion

Rll0 t o f u s i o n w e r e p r i m a r i l y seen a m o n g these latter MAbs. F o r e x a m p l e , M A b s t o t h e a l l o a n t i g e n c h a r a c t e r i z e d b y AMS 22.1 w e r e q u i t e c o m m o n in S J L a n t i - B A L B / c fusions, b u t M A b s t o c o r r e s p o n d i n g a l l o a n t i g e n s have n o t b e e n seen in o t h e r fusions. I t is also o f interest t h a t a l t h o u g h spleen cells w e r e u s e d as i m m u n o g e n s , M A b s t o T cell a l l o a n t i g e n s w e r e n e v e r f o u n d . T h e r e a s o n f o r this v a r i a b i l i t y is n o t u n d e r s t o o d . M a n y f a c t o r s can i n f l u e n c e t h e g e n e r a t i o n o f a n t i b o d i e s to alloantigenic specificities. I t is possible to g e n e r a t e A b t o d i f f e r e n t sets o f specificities d e p e n d i n g u p o n t h e n u m b e r , t i m e , and m o d e o f i m m u n i z a t i o n using t h e s a m e strains o f mice. T o e f f e c t i v e l y prod u c e A b s against a l l o t y p e s o f cell s u r f a c e i m m u n o g l o b u l i n s a p p e a r s t o r e q u i r e t h a t t h e d o n o r a n d r e c i p i e n t strains o f m i c e d i f f e r w i t h r e s p e c t to H-2 as well as a l l o t y p e ( H e r z e n b e r g a n d H e r z e n b e r g , 1978). This m a y also p r o v e t r u e f o r o t h e r ( m i n o r ) cell surface alloantigens. Since wells can b e r a p i d l y s c r e e n e d b y f l o w c y t o m e t r y t h e r e is n o n e e d t o r e s t r i c t t h e g e n e r a t i o n o r i d e n t i f i c a t i o n o f M A b s b y t h e choice o f m o u s e strains used f o r i m m u n i z a t i o n or as t a r g e t s in screening. I n e a c h fusion, e v e r y M A b g e n e r a t e d against a cell surface antigen is identified a n d partially c h a r a c t e r i z e d w i t h i n 2 days. U n i m p o r t a n t M A b s are d i s c a r d e d a n d t h o s e t h a t a p p e a r i n t e r e s t i n g can be i m m e d i a t e l y c l o n e d t o select stable p r o d u c i n g cell lines. T h e m u l t i p l e p a r a m e t e r analysis o f s a m p l e s allows t h e i d e n t i f i c a t i o n o f M A b s w h i c h b i n d t o as f e w as 5 - - 1 0 % o f t h e cells in a s a m p l e . T h e s e f a c t o r s m a k e f l o w c y t o m e t r y an o p t i m a l m e t h o d b y w h i c h M A b s t o n e w a n d / o r rare cell s u r f a c e antigens c a n be identified. ACKNOWLEDGEMENTS Supported by National Institutes of Health Grants AI-14782, CA-19266 a n d a g r a n t f r o m t h e Bristol M e y e r s F o u n d a t i o n . M.R.L. is a r e c i p i e n t o f Research Career Development Award AI-00348. REFERENCES Black, S.J., J.W. Goding, B.A. Gutman, L.A. Herzenberg, M.R. Loken, B.A. Osborne, W. Van der Loo and N.L. Warner, 1978, Immunogenetics 7,213. Bonner, W.A., H.R. Hulett, R.G. Sweet and L.A. Herzenberg, 1972, Rev. Sci. Instrum. 43,404. Brunsting, A. and P.F. Mullaney, 1974, Biophys. J. 14,439. Crosland-Taylor, P.J., 1953, Nature 171, 37. Dessner, D.S. and M.R. Loken, 1981, Eur. J. Immunol. 11, 282. Gohde, W., 1973, in: Fluorescence Techniques in Cell Biology, eds. A.A. Thaer and M. Sernetz (Springer, Berlin) p. 79. Green, M.C., 1979, Immunogenetics 9,197. Hammerling, G.J., B.D. Deak, G. Mauve, U. Hammerling and H.O. McDevitt, 1974, Immunogenetics, 1, 69. Heibert, R.D., 1979, in: Flow Cytometry and Sorting, eds. M.R. Melamed, P.F. Mullaney and M.L. Mendelsohn (Wiley, New York) p. 623. Herzenberg, L.A. and L.A. Herzenberg, 1978, in: Handbook of Experimental Immunology, ed. D.M. Weir (Blackwell Scientific Publication, Oxford) Ch. 12.

RIll

Herzenberg, L.A., R.G. Sweet and L.A. Herzenberg, 1976, Scient. Am. 234, 108. Herzenberg, L.A., L.A. Herzenberg, S.J. Black, M.R. Loken, K. Okumura, W. Van der Loo, B.A. Osborne, D. Hewgill, J.W. Goding, G. Gutman and N.L. Warner, 1977, Cold Spring Harbor Syrup. Quant. Biol. 41, 33. Hodkinson, J.R. and I. Greenleaves, 1963, J. Opt. Soc. Am. 53,577. Horan, P.K. and J.W. Kappler, 1977, J. Immunol. Methods 18,309. Horan, P.K. and L.L. Wheeless, 1977, Science 198,149. Julius, M.H., R.G. Sweet, C.G. Fathman and L.A. Herzenberg, 1975, in: Mammalian Cells: Probes and Problems, eds. C.R. Richman, D.F. Peterson, P.F. Mullaney and E.C. Anderson (ERDA Tech. Information, Oak Ridge, TN) p. 107. Kachel, V., 1979, in: Flow Cytometry and Sorting, eds. M.R. Melamed, P.F. Mullaney and M.L. Mendelsohn (Wiley, New York) p. 61. Kamentsky, L.A., M.R. Melamed and H. Derman, 1965, Science 150,630. Kaplow, L.S, 1965, Blood 26,215. Kaplow, L.S., 1979, in: Flow Cytometry and Sorting, eds. M.R. Melamed, P.F. Mullaney and M.L. Mendelsohn (Wiley, New York) p. 531. Kirker, M., 1969, The Scattering of Light and Other Electromagnetic Radiation (Academic Press, New York). Klein, J., 1975, Biology of the Mouse Histocompatibility-2 Complex (Springer, New York) p. 31. KShler, G. and C. Milstein, 1975, Nature 256,495. Krishan, A., 1975, J. Cell Biol. 66, 198. Laerum, O.D., T. Lindmo and E. Thorud, 1980, Flow Cytometry IV (Universitetsforlaget, Oslo). Leibson, P.J., M.R. Loken, S.J. Shapiro and H. Schreiber, 1980, Cancer Res. 40, 56. Loken, M.R., 1980a, Cytometry 1,136. L o k e n M.R., 1980b, J. Histochem. Cytochem. 28, 1136. L o k e n M.R. and L.A. Herzenberg, 1975, Ann. N.Y. Acad. Sci. 254, 163. Loken M.R. and D.W. Houck, 1981, J. Histochem. Cytochem. 29, 609. Loken M.R., R.G. Sweet and L.A. Herzenberg, 1976, J. Histochem. Cytochem. 24, 284. Loken M.R., D.R. Parks and L.A. Herzenberg, 1977a, J. Histochem. Cytochem. 25,790. Loken M.R., D.R. Parks and L.A. Herzenberg, 1977b, J. Histochem. Cytochem. 25,899. Loken M.R., R.D. Stout and L.A. Herzenberg, 1979, in: Flow Cytometry and Sorting, eds. M.R. Melamed, P.F. Mullaney and M.L. Mendelsohn (Wiley, New York) p. 505. Loken, M.R., D.S. Dessner-de Jose, G. Van Zant and E. Goldwasser, 1982, submitted for publication. Marshall, L., 1971, Laser Focus 7, 26. McKearn, T.J., F.W. Fitch, D.E. Smilek, M. Sarmiento and F.B. Stuart, 1979, Immunol. Rev. 47, 91. Melamed, M.R., P.F. Mullaney and M.L. Mendelsohn, 1979, Flow Cytometry and Sorting (Wiley, New York). Mie, G., 1908, Metallosungen. Ann. Physik. 25,377. Mullaney, P.F. and P.N. Dean, 1969, Appl. Optom. 8, 2361. Mullaney, P.F. and P.N. Dean, 1970, Biophys. J. 10, 764. Mullaney, P.F., M.A. Van Dilla, J.R. Coulter and P.N. Dean, 1969, Rev. Sci. Instrum. 40, 1029. Oi, V.L. and L.A. Herzenberg, 1980, in: Methods in Cellular Immunology, eds. B.B. Mishell and S.M. Shiigi (Freeman, San Francisco, CA) Ch. 17. Ozato, K. and D.H. Sachs, 1981, J. Immunol. 126, 317. Salzman, G.C., J.M. Crowell and C.A. Goad, 1975a, Clin. Chem. 21, 1297. Salzman, G.C., J.M. Crowell, J.C. Martin, T.T. Trujillo, A. Romero, P.F. Mullaney and P.M. LaBaure, 1975b, Acta Cytol. 19, 344. Salzman, G.C., P.F. Mullaney and B.J. Price, 1979, in: Flow Cytometry and Sorting, eds. M.R. Melamed, P.F. Mullaney and M.L. Mendelsohn (Wiley, New York} p. 105.

Rl12 Spectra-Physics Instruction Manual, 1976 (Spectra-Physics, Mountain View, CA) p. 1. Steen, H.B., T. Lindmo and O. Sorensen, 1980, in: Flow Cytometry IV, eds. O.D. Laerum, T. Lindmo and E. Thorud (Universitetsforlaget, Oslo) p. 31. Steinkamp, J.A., M.J. Fulwyler, J.R. Coulter, R.D. Heibert, J.L. Horney and P.F. Mullaney, 1973, Rev. Sci. Instrum. 44, 1301. Stohr, M., 1976, Cytophotometry 2, 39. Taylor, R.B., P.H. Duffus, M.C. Raff and S. De Petris, 1971, Nature New Biol. 233, 225.