Direct fluorescent labeling of cells with fluorescein or rhodamine isothiocyanate. I. Technical aspects

Journal oflmmunological Methods, 37 (1980) 97--108 © Elsevier/North-Holland Biomedical Press

97

DIRECT FLUORESCENT LABELING OF CELLS WITH FLUORESCEIN O R R H O D A M I N E I S O T H I O C Y A N A T E . I. T E C H N I C A L A S P E C T S '

EUGENE C. BUTCHER : and IRVING L. WEISSMAN a

Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. (Received 12 February 1980, accepted 1 May 1980)

A rapid and simple method of cell labeling by stable conjugation with fluorescein or rhodamine is described. Viable cells are incubated under benign conditions (near physiologic pH in normal media) with free fluorescein or tetramethyl rhodamine isothiocyanate, and are adequately separated from unreacted fluorochrome by washing or centrifugation through fetal calf serum. The effects of the pH, the time and temperature of incubation, and the concentration of cells, fluorochrome, and free protein in the media are described. The method labels all cell types, although to different degrees. Fluorescence microscopy reveals fluorescence throughout the cell, although chromatin appears relatively spared. Cellular fluorescence is fairly stable at 4 and 25°C, decays rapidly at 37°C, but is nonetheless visible for days even at this temperature. In the case of lymphocytes, intense fluorescence is obtained without affecting cell viability, and without alteration of the ability to mount a graft versus host response.

INTRODUCTION T h e m o s t c o m m o n l y e m p l o y e d t e c h n i q u e s f o r labeling cells utilize radioactive markers. These m e t h o d s lend themselves to a u t o m a t e d c o u n t i n g o f large n u m b e r s o f cells, b u t in general are less t e c h n i c a l l y s a t i s f a c t o r y w h e n applied to small cell n u m b e r s , are p o t e n t i a l l y b i o h a z a r d o u s , and pose difficult p r o b l e m s o f r a d i o a c t i v e waste disposal. F u r t h e r m o r e , their usefulness as cell m a r k e r s is limited since labeled cells c a n n o t be individually identified while viable. A stable f l u o r e s c e n t label f o r viable cells w o u l d p e r m i t s i m u l t a n e o u s i d e n t i f i c a t i o n and m o r p h o l o g i c and p h e n o t y p i c analysis o f individual m a r k e d cells, and y e t w o u l d also allow rapid e x a m i n a t i o n o f large cell n u m b e r s t h r o u g h flow c y t o f l u o r i m e t r y . Such a m e t h o d c o u l d facilitate, f o r instance, t h e s e p a r a t i o n o f r e s p o n d e r and target l y m p h o c y t e s in t h e f l o w c y t o m e t r i c analysis o f l y m p h o c y t e c y t o t o x i c i t y ; it w o u l d p e r m i t use o f f l u o r e s c e n t internal s t a n d a r d or internal c o n t r o l p o p u l a t i o n s in t h e 1 Supported by U.S.P.H.S. Grant AI 09072. : To whom reprint requests should be sent. Recipient of NIH Postdoctoral Training Grant, GM 002236-04 in Experimental Pathology. 3 Faculty Research Awardee of the American Cancer Society.

98 analysis of cellular interactions; and it could be used to trace the fate of various types of cells in vivo in animals and humans. Most previous reports of cell labeling with fluorescent c o m p o u n d s have described the use of materials which are either actively taken up by cells (e.g. fluorescein diacetate: R o t m a n and Papermaster, 1 9 6 6 ) o r interact with cellular nucleic acids (acridine related compounds: De Bruyn et al., 1950; Farr, 1951; White, 1954; Desai and Creger, 1963). In our experience, these substances are unsuitable for cell tracer experiments because the label is rapidly reversible, or is taken up excessively b y unlabeled bystander cells. For this reason we wished to investigate the use of fluorochromes that could be covalently linked to cell proteins. Although other reactive fluorochromes have been described that might be useful in this c o n t e x t (e.g. bimanes: Kosower et al., 1979), we chose to study the isothiocyanate derivatives of fluorescein and rhodamine because of their general availability and stability. The isothiocyanate group on these molecules is thought to react preferentially with the epsilon-amino nitrogen of lysine (see Nairn, 1976), and thus they would be expected to label most proteins. Rhodamine isothiocyanate (RITC) has been previously used as a fluorescent probe of l y m p h o c y t e activation (Nairn et al., 1979) b u t the labeling method described by these authors was harsh (pH 8.6 buffer, following by dialysis of labeled cells against activated charcoal). Fluorescein isothiocyanate (FITC) has been used to modify the antigenicity of the cell surface of lymphocytes (Friedman et al., 1979), again using unphysiological labeling conditions (pH 9.0). We present an analysis of (1) factors affecting l y m p h o c y t e labeling with FITC and RITC, and (2) the effect of labeling on cell viability and functional capacity. This analysis defines a stable, rapid, simple and inexpensive method of in vitro cell labeling under mild conditions. MATERIALS AND METHODS

Animals BALB/c.H-2 b and (C57BL/6J X BALB/c) F1 and F2 hybrids were bred and maintained in our colony.

Medium Standard cell suspending medium (CSM) was 5% fetal calf serum in equal parts of Medium 199 (Grand Island Biological Company) and Dulbecco's phosphate buffered saline (PBS), pH 7.4.

Preparation of lymphocyte suspensions Minced organs were pressed gently through 200 gauge stainless steel screen and suspended in CSM. Cell viability was assessed by exclusion of trypan blue or propidium iodide.

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Preparation o f fluorescein isothiocyanate and rhodamine isothiocyanate stock solutions Stock solutions of 600 pg/ml fluorescein isothiocyanate (FITC, Isomer I, Sigma Chemical Company, St. Louis, MO) and 30 pg/ml tetramethyl rhodamine isothiocyanate (RITC, Isomer R, BBL, Cockeysville, MD) in PBS were prepared b y shaking an excess of FITC or RITC crystals in PBS for 1--3 h at room temperature and removing the undissolved crystals by centrifugation (1500 X g, 15 min) and filtration through Millipore filters (0.22 pm pore size). The resulting concentrations were determined spectrophotometrically using estimated molar extinction coefficients of 68,000 at 495 nm in PBS for FITC, and 70,000 at 550 nm in 70% ethanol for RITC. The stock solutions were stored in aliquots at --35°C until use. The molecular weight of FITC is a b o u t 389, and that of RITC is 444.

Cell labeling with FITC or RITC Cell labeling was routinely carried out with 2.5--5 X 107 cells/ml for 10--20 min. Labeling with RITC was always performed at room temperature (RT) in normal CSM (pH 7.4). Labeling with FITC was performed at 37°C in CSM, or at RT in CSM adjusted to pH 7.0 with HC1. The concentration of reactive fluorochrome was varied according to the degree of fluorescence required. After incubation, labeling was stopped b y adding cold medium, and labeled cells were separated from reactive fluorochrome by centrifugation through a large (at least 5 cm in depth) cushion of fetal calf serum. They were then either resuspended in CSM for further experimental manipulation, or in 1% formaldehyde in slightly hypertonic (1.25X normal concentration) PBS, pH 7.4, for analysis on the fluorescence-activated cell sorter.

Fluorescence microscopy A Zeiss microscope equipped with HBO 50 W mercury vapor lamp epiillumination and exciter/barrier filter combinations for rhodamine (546.1 nm excitation) and fluorescein (440--490 nm excitation) was used.

Quantitation of fluorescence with the fluorescence activated cell sorter (FAGS) Analyses were carried o u t on a Becton Dickinson FAGS III equipped with a 2 W laser (Model 164 Spectra-Physics) operated at 400 mW. Excitation was at 488 nm, and the standard cut on filters supplied by Becton Dickinson for use with fluorescein were employed (520 nm interference type, and 530 nm absorbing glass, Detric Optics, Inc., Marlboro, MA). In this paper, one unit of fluorescence is arbitrarily defined as 1/18 the modal fluorescence of glutaraldehyde fixed chicken red blood cells (CRBC),

100 which provide a convenient and adequately stable fluorescence standard when prepared and stored as described by Herzenberg and Herzenberg (1978). With the filter combination used, the modal fluorescence of fixed CRBC is approximately equivalent to 106 molecules of FITC (M. Loken, personal communication). To avoid the necessity of frequently readjusting the FACS, the same filter and excitation combination described above, which is optimal for fluorescein, was also used for analysis of rhodamine. Thus, the units of fluorescence of rhodamine and fluorescein are not directly comparable in terms of usefulness or subjective brightness. All fluorescence analyses were done on lymphocytes that were viable at the time of fixation. Dead cells were exclt/ded by their light scattering properties (see Herzenberg and Herzenberg, 1978).

Suspension staining for identification of T and B cells Cell suspensions were stained using standard techniques with rhodaminated rabbit anti-mouse immunoglobulin antiserum, or with a first stage monoclonal rat antibody against the Thy 1 antigen (31--11, kindly provided by Eric Pillemer in our laboratory) followed by a highly specific rhodaminated rabbit anti-rat immunoglobulin second stage.

Lysis of plasma membranes Manual lysing agent (B3145-6A, Scientific Products) was used to lyse l y m p h o c y t e plasma membranes. RESULTS

(I) Conditions of labeling In a series of experiments designed to define the factors affecting the degree of conjugation of FITC or RITC to cells, lymphocytes were incubated with the reagents under controlled conditions and each of the important elements of the incubation were varied individually. Marked differences in the labeling properties of FITC and RITC were defined in these experiments. The experimental conditions are summarized in the legend to Fig. 1, which presents these results. Concentration of reactive label. Cellular fluorescence increases with increasing concentrations of FITC and RITC; fluorescence is directly proportional to RITC concentration over the range examined (0.5--17 pg/ml), but rises more rapidly than the concentration of FITC over the range of 0---60 pg/ml (Fig. 1A). pH. Alterations in the pH of the incubation mixture in the range 6.8-7.8 had no effect on labeling with RITC, whereas cellular labeling with FITC

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Fig. 1. The effect o f various factors on cell labeling with FITC and RITC. Each of the important elements of the incubation were varied individually under otherwise standard labeling conditions. Standard conditions of incubation were 2.5 × 107 cells/ml in m e d i u m composed o f 5% fetal calf serum in an equal mixture of m e d i u m 199 and PBS, pH 7.4, for 20 min at 37°C with 69 pg/ml FITC, or for 15 min at room temperature with 3.4 pg/ml RITC. Figures show the effect on cellular fluorescence of variation in: (A) concentration of reactive fluorochrome; (B) pH (adjusted with 1 M acetic acid or sodium hydroxide); (C) time o f incubation; (D) temperature; (E) concentration of cells; (F) concentration of protein (fetal calf serum) in the medium.

decreased markedly with increasing pH over the same range (Fig. 1B). This dependence of FITC labeling was also observed in the absence of protein in the medium (data not shown). Time. Cellular fluorescence increases rapidly with time after addition of reactive fluorochrome but reaches a plateau at about 20 min (Fig. 1C). In the case of FITC, cellular fluorescence then begins to decline slowly, presumably because of turnover of reacted label at 37 ° C. The plateau is pre-

102 dominately due to depletion of reactive fluorochrome rather than saturation o f b i n d i n g sites i n t h e cell, s i n c e a d d i t i o n o f m o r e l a b e l c a u s e s a f u r t h e r r a p i d i n c r e a s e i n c e l l u l a r f l u o r e s c e n c e o f n e a r l y t h e s a m e m a g n i t u d e as t h e i n i t i a l labeling step. "o

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Fig. 2. Fluorescence activated cell sorter scatter plots of the distribution of fluorescence among labeled lymphocytes. In each figure, the abscissa represents cellular fluorescence and the ordinate is low angle light scatter, a property that is related to cell size and viability• Each dot represents a single cell. (A) RITC: viable lymphocytes (L) appear to be labeled with RITC in relation to cell size, since they constitute a single group of cells in which fluorescence increases with light scatter• Dead cells (D). (B) FITC: viable FITClabeled lymphocytes are divided into two discrete populations• The brighter group is predominantly B cells (B), and the dimmer group is mostly T cells ( T - see text)• Dead cells (D). Contaminating red blood cells, the small cluster of cells in the lower left hand corner, are labeled much less brightly than viable lymphocytes.

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Temperature. Labeling with both reagents is influenced by the temperature of incubation, b u t the effect was more marked with FITC than RITC. FITC label was only 1/3 as bright at 25°C, and only 3% as bright at 0°C as at 37°C (Fig. 1D). Concentration o f cells. The fluorescence associated with each cell after RITC labeling decreases progressively with increasing cell concentration in the incubation mixture; the intensity at l 0 s cells/ml is only 43% of that at 5 X 106 cells per ml. FITC labeling, on the other hand, is little affected b y cell concentration under the conditions used (Fig. 1E). Concentration o f free protein. Since fetal calf serum (FCS) is generally added to cell suspending media in this laboratory, we examined its effect on cell labeling. Increasing concentrations of FCS from 0.2 to 50% in the incubation mixture progressively inhibited cell labeling with both FITC and RITC, although the inhibition of FITC labeling was somewhat greater (Fig. 1F). (II) Labeling o f different cell types Although FITC and RITC will label any cell, different types of cells are labeled to different degrees. Mouse red blood cells, for instance, label much less brightly than lymphocytes. The primary determinant of the extent of labeling of the given cell type may well be its protein content, although other variables such as membrane permeability or internal pH could also play a role. Certainly, there is in general a positive correlation between cell volume and fluorescence intensity within a given cell type. (Whether fluorescence and volume are directly proportional or not is not known, since our justification for this statement is the correlation of fluorescence with low angle light scatter in the FACS -- see Fig. 2A -- which itself is related to b u t not linear with cell volume (Loken and Herzenberg, 1975).) Surprisingly, FITC distinguishes between two classes of peripheral lymphocytes, as shown in the scatter plot presented in Fig. 2B. These two classes prove to be B cells and T cells; splenic lymphocytes sorted with the FACS from the brighter group were predominantly B cells (86% immunoglobulin positive, 9% T h y 1 positive), whereas those from the dimmer group were predominantly T cells (89% Thy 1 positive, 6% immunoglobulin positive). RITC apparently does not separate these two subclasses, since FACS analysis of RITC labeled l y m p h o c y t e s shows a single group of cells in which fluorescence increases with cell volume (scatter) (Fig. 2A).

(III) FITC and RITC bind to internal cell components Photographs of FITC- and RITC-labeled monolayers of malignant fibroblasts (Fig. 3) clearly demonstrate that both cytoplasmic and nuclear elements are labeled. We presume that membrane components are also labeled. Chromatin appears relatively spared. In the case of lymphocytes, the vast majority of label is internal: over 70%

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Fig. 3. Fibroblast monolayers labeled with RITC (A) or FITC (B) and washed. fluorochromes label both cytoplasmic (c) and nuclear (n) elements. Chromatin, e identified in mitotic figures (m), appears relatively spared.

o f t o t a l cellular f l u o r e s c e n c e is associated with t h e nucleus, as d e t e r m i b y d i r e c t c o m p a r i s o n o f f l u o r e s c e n c e on the FACS b e f o r e and a f t e r ] o f plasma m e m b r a n e s .

(IV) Decay of fluorescence of labeled cells One i m p o r t a n t f e a t u r e o f a cell m a r k e r is its stability with time. As sh~ in Fig. 4, the f l u o r e s c e n c e o f b o t h F I T C and R I T C labeled cells was f~ stable at 4°C, and d e c a y e d to half its initial value b y 18 h at 25°C. A t 3: t h e r e was an i m m e d i a t e rapid d e c a y (half-time r o u g h l y 1 h), to a b o u t ', o f initial f l u o r e s c e n c e , f o l l o w e d b y a m u c h slower decline t o 15--20~, initial f l u o r e s c e n c e at 18 h. In e x p e r i m e n t s in w h i c h labeled cells v injected in vivo i n t o syngeneic recipients, the same rapid initial d e c a y is s, nonetheless, cells m a i n t a i n a slowly d e c a y i n g (half-life a b o u t 3 days)

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significant and usable level of fluorescence for days (see accompanying paper).

(V) Transfer o f fluorescence from labeled to unlabeled cells When RITC-labeled lymphocytes are mixed with unlabeled lymphocytes, the latter also become fluorescent, although not to the same level. Microscopically, the distribution of fluorescence in these cells is indistinguishable from that in directly labeled cells. Transfer of fluorescence (per unlabeled cell) increases with the concentration of labeled cells, the ratio of labeled to unlabeled cells, and the time of co-incubation (data not shown). Extensive washing of labeled cells decreases transfer of label to some extent, as does incubation of labeled cells at 37°C in protein-containing medium prior to mixing with unlabeled ones. These observations suggest that a significant a m o u n t of unreacted RITC may remain associated with labeled cells after simple washing. In striking contrast, no significant transfer of fluorescence is observed from FITC-labeled cells even after minimal washing.

(VI) Effect o f label on cell viability and function We have never observed a significant loss of cells or an immediate decrease in cell viability after labeling with RITC or FITC (even at the highest levels employed in the experiment in Fig. 1).

106 TABLE 1 Comparison of the ability of labeled and unlabeled lymphocytes to mount a graft vs. host response a. Recipient

Donor cells

Label

BALB/c x C57BL)F1 BALB/c x C57BL)F1 BALB/c X C57BL)F1

(BALB/c x C57BL)F1 (BALB/c X C57BL)F1 (BALB/c x C57BL)F1

None RITC c FITC d

BALB/c X C57BL)F1 BALB/c x C57BL)F1 BALB/c x C57BL)F1

BALB/c. H-2 b BALB/c. H-2 b BALB/c. H-2 b

None RITC FITC

PLN index b 4.2 -+ 0.5 3.3 -+ 0.3 4.7 -+ 0.6 15.1 -+ 2.7 14.0 -+ 3.5 21.9 -+ 1.0

a 1.5 X 107 viable unlabeled or labeled lymph node cells in 0.25 ml CSM were injected into the left footpad of recipients, with three recipients per sample. 7 days later, the left and right popliteal lymph nodes (PLN) were weighed. There was no significant difference in the average weights of the right (uninjected) PLN of the various groups. Lymphocyte donors were 3--4 months old; recipients were 4--6 weeks old. b The ratio of the mean weight of the left popliteal lymph nodes (PLN, injected side) of the three recipients to the average weight of the right PLN (uninjected side). c 3.0 pg/mt RITC, 5 x 107 cells/ml, 10 min at room temperature in CSM. Median fluorescence 24--25 units. d 60 pg/ml FITC, 5 x 107 cells/ml, 20 rain at 37°C in CSM. Median fluorescence 300-320 units. As a simple test o f the f u n c t i o n o f labeled l y m p h o c y t e s , we selected t h e graft versus h o s t response as m e a s u r e d b y the popliteal l y m p h n o d e assay, w h i c h d e p e n d s o n cell viability, i m m u n e responsiveness, and the c a p a c i t y o f d o n o r cells to divide (see D o r s c h and Roser, 1974). As s h o w n in Table 1, at the levels o f labeling used in this e x p e r i m e n t , t h e ability o f labeled 'parental' cells to m e d i a t e the r e s p o n s e was at least as great as t h a t o f unlabeled cells. (The labeling p r o t o c o l s used in this e x p e r i m e n t , described in the table f o o t n o t e s , are s t a n d a r d in this l a b o r a t o r y . T h e y were selected because the degree o f labeling t h e y yield does n o t significantly alter l y m p h o c y t e h o m i n g in vivo (see a c c o m p a n y i n g paper).) DISCUSSION T h e r e are striking d i f f e r e n c e s b e t w e e n the cell labeling characteristics o f F I T C and R I T C . These d i f f e r e n c e s are p r o b a b l y a result o f charge d i f f e r e n c e s u n d e r t h e c o n d i t i o n s o f labeling, w h i c h d e t e r m i n e their ability to cross t h e p l a s m a m e m b r a n e and label intracellular proteins. Over t h e entire pH range t e s t e d , R I T C is a n e u t r a l m o l e c u l e . (The t e r t i a r y amines [Fig. 5] w o u l d n o t be e x p e c t e d to a c c e p t p r o t o n s at n e u t r a l pH because o f r e s o n a n c e stabilization.) Thus, R I T C can freely equilibrate across the cell m e m b r a n e , and the degree o f cell labeling is t h e r e f o r e i n d e p e n d e n t o f extracellular pH. T h e neutral, relatively lipophilic c h a r a c t e r o f R I T C m a y also explain t h e

107

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NCS

Fig. 5. The structure of FITC and RITC. tendency of RITC-labeled cells to transfer fluorescence to unlabeled cells even after extensive washing: RITC may associate noncovalently with cell lipids or hydrophobic proteins, or be sequestered in active form in the cell membrane lipid bilayer (see also Nairn, 1976). FITC has h y d r o x y l groups in place of the tertiary amines of RITC (Fig. 5), and these h y d r o x y l groups can be ionized. Fluorescein, a closely related molecule that differs from FITC only in lacking the reactive isothiocyanate moiety, has a pK1 of 4.3 and pK2 of 6.7 (Abdel-Halim et al., 1970). Thus, FITC is negatively charged near physiologic pH, and in fact is probably in equilibrium between the singly and doubly ionized forms, with the latter predominating. Only one in a thousand FITC molecules will be neutral at pH 7.3. We do not know whether singly ionized FITC can enter the cell, or if penetration of the membrane depends on the rare neutral forms. Nonetheless, it is apparent that these neutral and singly ionized species are favored at lower pH. Thus, we propose that cells are more brightly labeled at lower pH because more FITC can penetrate the cell membrane. (It should be emphasized that, since the internal pH of viable cells is actively maintained, variations in external pH are not expected to affect the labeling efficiency of reactive molecules that are within the cell. Since cellular fluorescence appears to be mostly internal, it follows that, if limiting, the ability of the reactive fluorochrome to gain entry into the cell would be the primary determinant of fluorescence. This interpretation is supported by the absence of affect of pH on RITC labeling.) The requirement for penetration of the cell membrane may also explain the somewhat increased temperature dependence, and the greater inhibition by extracellular protein of FITC- as opposed to RITC-labeling. We have no ready explanation for the t e n d e n c y of FITC but not RITC to label preferentially B lymphocytes, although it is interesting to speculate that it may reflect differences in membrane composition and permeability, or in intracellular pH. The differences between FITC and RITC may determine or limit their usefulness as cell labels for particular applications. For instance, transfer of fluorescence from RITC-labeled cells may restrict the usefulness of this reagent to situations in which the labeled cells are a small minority, or are present in fairly low concentrations, such as in tracing labeled cells after injection in vivo. We have presented evidence that cell labeling with these fluorochromes is remarkably benign. Labeling does not alter immediate cell viability, nor

108 d o e s it i n t e r f e r e w i t h the cell f u n c t i o n s we have m e a s u r e d . Even heavily labeled l y m p h o c y t e s m a i n t a i n t h e ability to m o u n t a g r a f t versus h o s t r e s p o n s e and t h u s are n o t o n l y i m m u n o l o g i c a l l y c o m p e t e n t b u t are pres u m a b l y c a p a b l e o f dividing n o r m a l l y , as well (see D o r s c h and Roser, 1974). It s h o u l d be stressed, h o w e v e r , t h a t labeling is n o t e n t i r e l y w i t h o u t effect. F o r instance, l y m p h o c y t e s excessively labeled w i t h F I T C or R I T C d o n o t h o m e n o r m a l l y to l y m p h o i d tissues in vivo (see a c c o m p a n y i n g p a p e r ) . Thus, as w i t h o t h e r labeling t e c h n i q u e s , it is i m p o r t a n t t h a t t h e degree o f labeling be t i t r a t e d to t e s t f o r adverse e f f e c t s o n t h e biological f u n c t i o n b e i n g tested. In c o n c l u s i o n , cell labeling b y c o v a l e n t i n t e r a c t i o n w i t h F I T C or R I T C is rapid, simple, i n e x p e n s i v e , and r e m a r k a b l y benign. We have p r e v i o u s l y r e p o r t e d t h e successful use o f F I T C a n d R I T C to p r o v i d e a f l u o r e s c e n t i n t e r n a l s t a n d a r d p o p u l a t i o n in studies o f cell-cell a d h e s i o n ( B u t c h e r et al., 1 9 7 9 a , b, c), to label t h y m o c y t e s in situ as a m e a n s o f f o l l o w i n g t h e i r m a t u r a t i o n (Scollay et al., 1 9 7 8 ) , and as a cell m a r k e r in studies o f the in vivo m i g r a t i o n o f n e o p l a s t i c l y m p h o c y t e s (Warnke et al., 1979). We feel t h a t this m e t h o d o f labeling cells w i t h stable f l u o r e s c e n c e will p r o v e to be a p p l i c a b l e to m a n y p r o b l e m s in cellular i m m u n o l o g y a n d cell b i o l o g y in general. ACKNOWLEDGEMENTS We t h a n k S. J a c o b s , J. Cockriel, M. Beers and D. C h e s t n u t f o r t h e i r able assistance, and W. Huestis f o r her h e l p in u n d e r s t a n d i n g t h e c h e m i s t r y o f fluorescein. REFERENCES Abdel-Halim, F.M., R.M. Issa, M.S. EI-Ezaby and A.A. Hasanein, 1970, Z. Phys. Chem. 73, S.59. Butcher, E.C., R.G. Scollay and I.L. Weissman, 1979a, Adv. Exp. Med. Biol. 114, 65. Butcher, E.C., R.G. Scollay and I.L. Weissman, 1979b, Nature 280, 496. Butcher, E.C., R.G. Scollay and I.L. Weissman, 1979c, J. Immunol. 123, 1996. De Bruyn, P.P.H., R.C. Robertson and R.S. Farr, 1950, Anat. Rec. 108,279. Desai, R.G. and W.P. Creger, 1963, Blood 21,665. Dorsch, S.E. and B. Roser, 1974, Aust. J. Exp. Biol. Med. Sci. 52, 253. Farr, R.S., 1951, Anat. Rec. 109,515. Friedman, S.M., O. Irigojen, J. Kuhns and L. Chess, 1979, J. Immunol. 123,496. Herzenberg, L.A. and L.A. Herzenberg, 1978, in: Handbook of Experimental Immunology, 3rd Edition, ed. D.M. Weir (Blackwell Scientific Publications, Oxford) p. 22.6. Kosower, N.S., F.M. Kosower, G.L. Newton and H.M. Ranney, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 3382. Loken, M.R. and L.A. Herzenberg, 1975, Ann. N.Y. Acad. Sci. 254,163. Nairn, R.C., 1976, Fluorescent Protein Tracing, 4th Edition (Churchill-Livingstone, Edinburgh). Nairn, R.C., I.M. Jablonka, J.M. Rolland, G.M. Haltiday and H.A. Ward, 1979, Immunology 36, 235. Rotman, B., and B.W. Papermaster, 1966, Proc. Natl. Acad. Sci. U.S.A. 72, 1231. Scollay, R., M. Kochen, E. Butcher and I. Weissman, 1978, Nature 276, 79. Warnke, R.A., S. Slavin, R.L. Coffman, E.C. Butcher, M.R. Knapp, S. Strober and I.L. Weissman, 1979, J. Immunol. 123, 1181. White, L.P., 1954, Blood 9, 73.