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Automated scanning of bone marrow cell colonies growing in agar-containing glass capillaries
Experimental Cell Research 103 (1976) 271-277
AUTOMATED SCANNING OF BONE MARROW CELL COLONIES GROWING IN AGAR-CONTAINING GLASS CAPILLARIES H. R. MAURER and R. HENRY Pharmazeutisches Institut der Freien Universitiit Berlin, D-1000 Berlin 33, Germany
SUMMARY An optical scanning system was developed to determine the growth of clusters and colonies of granulocytes and macrophages from mouse bone marrow cells in agar capillary tubes. The system consists of a commercially available photometer with a densitometer attachmen:, a two-mirror set to receive the light scattered by the cell colonies, a multiple capillary holder and an automatic sample changer. Parameters affecting scanning were examined and optimized: background scatter. instrument adjustments (e.g. signal damping) and threshold settings for clusters and colonies. Combined with the advantageous agar capillary technique, the complete scanning system provides an easy, accurate and sensitive method for rapid quantitation of hemopoietic cell colony formation in vitro.
The in vitro agar culture technique, originally introduced by Pluznik & Sachs [l] and Bradley & Metcalf [2], has had a large impact on present concepts of the proliferation and differentiation of hemopoietic progenitor cells. The visual examination of a great number of colonies as usually cultivated in Petri dishes, may become tedious and uneconomical when many Petri dishes have to be scored. This problem arose when we used the method to test for specific inhibitors (chalones) of granulopoiesis separated by biochemical techniques [3-51. We therefore adopted a modification of the agar colony method using glass capillary tubes [6] and studied in detail the parameters affecting the growth of mouse bone marrow cells in this system [7]. Its main advantage, amongst others, is that the colonies can be easily scanned on the basis of their light-scattering properties. However, the electronic counting, as performed by
Abrams et al. [6], requires photoelectric scanning equipment which is not commercially available. Upon our suggestion, Zimmer & Neuhoff [8] found a simple yet elegant way to adapt available photodensitometers to this purpose by means of a twomirror system. To scan ten capillary samples in sequence we have developed an automatic multiple sample changer that can be loaded with a multiple tube holder. This report describes the complete arrangement and the requirements for accurate and reproducible cluster and colony scanning. The various advantages offered by our automated technique will be demonstrated elsewhere 19, 101. MATERIALS
AND METHODS
Cell culture techniques The method of the in vitro agar culture of mouse bone marrow cells in glass capillary tubes of 1.38 mm internal diameter has been described in detail [?‘I. Meuse
272
Maurer and Henry Scattoreci
Pen recorder
Disc
light
dtmhment .?K
5
embryo conditioned medium was used as the source of colony stimulating activity in preference to mouse endotoxin-stimulated serum. Use of the former produced colonies of purely granulocytic cells whereas the latter gave colonies of highly vacuolized, large cells which were extremely difficult to identify. The preparation of mouse embryo conditioned medium is described elsewhere [9].
Scanning instruments We used a PMQ3 Photometer System with an automatic amplification unit, together with the ZK5 disc gel scanning attachment (Carl Zeiss, Oberkochen, Germany). The scanning attachment was adapted for light scattering measurements by use of two mirrors [8], and a built-in automatic sample changer enabled us to scan ten capillaries consecutively (fig. 1). The analog output of the amplifier unit was connected to a Servogor S, Type RE 541, linear potentiometric flat bed pen recorder (Goerz Electra GmbH, Vienna). The various settings of the scanning system, e.g. amplification slit width, damping, were adjusted to produce a pen deflection of about 90 7%when scanning the largest colonies grown in the agar gels, whilst retaining enough sensitivity to determine smaller cell clusters. We preferred the following settings for most scannings: PMQ3, high voltage amplification step 2, automatic amplification 10x, damping step 2, output voltage 10 mV, slit width 0.3 mm, wavelength 515 nm; ZKS, scan speed 30 mmlmin; Servogor S, recorder speed 60 mmlmin, sensitivity 20 mV.
Photometer PM0 3
system
so on. When the last, tenth capillary has been scanned, microswitch 2 reverses the direction of movement of the drive shaft and the holder returns to zero position. Microswitch 3 terminates the programme in zero position. The electronics controlling these movements are “grafted” onto the ZK5; thus this instruments can be still used for other scanning purposes. The time required to scan a capillary containing 100 pl of gel amounted to 3 min at a speed of 30 mm/mm of the ZK5, and the programme is completed in about 45 min.
RESULTS Fig. 1 shows the complete instruments system for scanning bone marrow cell colonies growing in agar contained in glass capillaries. Monochromatic light of optimal wavelength of 515 nm (fig. 2) is passed through the glass capillary moving through
6-
‘7
5-
/
When the programme is activated, the capillary holder with its ten glass capillary tubes moves from zero position in a horizontal direction through the light path thereby scanning the colonies in the first capillary. When the scan is complete a microswitch terminates the horizontal movement and induces a signal in the drive motor which lifts the capillary holder at 30 rpm 10 mm higher on a drive shaft. Microswitch 1 stops this movement and the second capillary is scanned and Exp
Cell
Res
103 (I 976)
/
‘A, ‘1.
4.
Mode of action of the automatic capillary changer (fig. 4)
Fig. 1. Scanning system for cell colonies growing in agar capillaries by light scattering. The system comprises a spectrophotometer (PMQ 3); a disc attachment (densitometer ZK 5) with built-in automatic capillary changer, an inserted multiple capillary holder and a twomirror set for light scattering detection; and a pen recorder. See Materials and Methods for details.
/
1.
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./
1.
r
\.
4@J
4&J
MO
S&O
600
650
Fig. 2. Abscissa: light wavelength (nm); ordinate: peak height (cm). Scanning granulocytic colonies in agar capillaries at different wavelengths of the monochromatic light. Monochromator slit width 0.3 mm. For other conditions see Materials and Methods.
Automated scanning of bone marrow cell coiofzles
%\ ‘Rubber
gasket
I
5cm
,
Fig. 3. Multiple capillary holder (made from Plexiglasj. See Materials and Methods for details.
the light path of the disc gel attachment ZK 5. The capillary is held in a multiple capillary rack (fig. 3) carried by the automatic sample changer (fig. 4). The direct light path is blocked by the blackened back of mirror 2 fixed at an angle of 45” relative to the optical axis. Light scattered by the colonies in the capillary at an angle of 3045” is reflected by mirror 1 and received by the photomultiplier via mirror 2. Both mirrors of about 40 mm diameter are fixed onto a support plate and adjusted for maximum sensitivity. A slit width of 0.3 mm was chosen to give the best results, a reasonably flat baseline and a good signal response to colonies in the light path. A smaller slit width also produces a good baseline but only small peaks, whilst a larger slit produces a poor baseline and poor resolution. The slit height must exceed the diameter of the capillary and, as the light above and below the capillary will not be scattered, it does not contribute to the signal. A multple capillary rack was constructed to hold ten capillary tubes in a fixed, parallel and horizontal position (fig. 3). The holder itself consists of two frames, one
273
with four rivets and ten grooves for the capillaries, the other with four rubber gaskets to hold the tubes fast and four bores provided for the rivets to hold both frames together. The capillary holder can be easily put together and dismantled and is used for both the total period of incubation and the scanning procedure, obviating the need of further capillary manipulation. For automated scanning of ten capillaries in sequence, the holder with the fixed tubes is inserted into the automatic mulliple sample changer (fig. 4). This device moves the holder in meander-like path through t light beam. Its horizontal drive is provided by the disc attachment ZK5. while the vertical movement is given by a drive motor built into the sample changer. The resolving capacity of the scanning system is demonstrated by the photograph of several colonies (fig. 5). Evidently the peak height mainly depends on the cell concentration per unit volume, whereas t
4. Automatic multiple capillary changer. See Materials and Methods for details. The numbers I-10 designate the holder positions, (l)-(2) the microswitches.
Fig.
274
Maurer and Henry
20
0t-
capillary section; ordinate: scanner (pulse) count. Capillaries filled with water, 0.18 % agar in medium without and with lo4 mouse bone marrow cells/l00 ~1 were scanned under identical scanning conditions (see Materials and Methods).
Fig. 6. Abscissa:
Fig. 5. Granulocytic colonies in 0.18% agar medium
contained in a glass capillary tube (1.38 mm i.d.) with recorder scan, Range of cell no./colony: 235-296. Photo: B. Kamann.
peak area is the product of cell concentration and colony diameter. Background scatter mainly results from surface scratches on the glass tubes, electrostatically charged dust particles on the glass surface, particles contained in the agar and cell debris. Fig. 6 shows that the agar itself raises the background level and may produce small artifact peaks which, however, will not “grow” and can thus be distinguished from cell colonies. The sensitivity and resolution of the scanning system can be used in several ways depending upon the requirements of the operator. For example, from fig. 7, selecting damping step 6 provides the best Exp CeNRes 103 (1976)
signal-to-noise ratio and should be preferred. Other relevant instruments settings include high voltage amplification, scan speed, recorder speed and recorder sensitivity. For a quick evaluation of colony growth the capillaries are scanned at maximum possible speed (30 mmlmin) and all peaks above the pre-set threshold level are counted as colonies. Only when a colony grows directly behind another, i.e. along the optical axis, it is not possible to register a correct count. However, by regulating the
gel section; ordinate: pulse count. Selecting the optimum damping step (a-c): Signal/ noise ratio versus signal damping. Colonies at day 7 from lo4 bone marrow cells seeded per capillary. Scanning conditions see Materials and Methods.
Fig. 7. Abscissa:
Automated scanning of bone marrow cell colonies
Wlcq
CO”“t
no. of signals (pulses); ordinate: signal height (cm). Threshold settings for clusters and colonies. Clusters and colonies at day 7 from 10’ bone marrow cells seeded per capillary. Scanning conditions see Materials and Methods.
Fig. 8. Abscissa:
number of cells seeded this rarely occurs on statistical grounds. When a daily growth curve is desired the capillaries may be slowly scanned, producing larger peaks of greater area which may be accurately measured. Can the scanner discriminate between clusters and colonies? Clusters are, arbitrarily, defined as an accumulation of about 5-50 cells, whereas colonies consist of more than about 50 cells. The question of threshold setting was approached by the graph shown in fig. 8. Plotting the number of signals from a capillary versus the signal height yielded a curve with a decreasing slope relative to the abscissa; its inflection point was taken as cluster level. Visual counting using a stereomicroscope led to the colony level. Selecting a particular threshold level thus provided a fairly good accordance between eye and machine counting of clusters and especially, of colonies (fig. 9). DISCUSSION The principal advantages of culturing hemopoietic cells in agar contained in glass capillary tubes have been discussed at length
275
[6, 71. The cells forming clusters and colonies in agar have previously been identified as granulocytes, macrophages and mixtures of both, depending on the colonystimulating factor that was used [II]. Our studies have further documented the main feature of the capillary technique, i.e. its applicability to optical scanning. Previous reports [6, 12-14 on the use of light scattering for colony scanning have not led to widespread use, probably because suitable instruments were not available. BWetrH, the simple two-mirror system [g] can be adapted to many commerciai densitometers. The use of a multiple capillary holder and of an automatic sample changer has substantially improved the technique allowing us to accurately scan a great number of capillaries. All sera, media and samples containing colony-stimulating activities should be filtered through 0.2 pm membranes before use to produce as optically clear gels as possible. Bacterial colonies are much larger than the granulocytic coionies and are.
Fig. 9. Abscissa: visual colony counl (no. of colonies and clusters); ordinate: scanner count (no. of peaks).
0-O. Colonv and cluster count at 1 cm threshold height (r=O.982. szy= 3~1.82); Cm, colony count at 2 cm threshold height (r=0.995. s,,,=+O.71). Visual versus icanner counting of clusters and colonies at preset threshold levels of 1 and 7 cm. Clusters and-colonies at day 7 from 10’ bone marrow cells seeded per capillary. Scanning conditions see Materials and Methods.
276
Maurer and Henry
however, very seldom observed due to sterile operating procedures. Bacterial and fungal contamination during incubation is readily detected by cloudy gel ends which when scanned produce very large and broad signals. Therefore, provided that sterile, clean liquids are used, it can be seen (figs 6, 7) that the only signals produced above background scatter are, normally, those arising from cells and colonies inside the agar gel, no optical interference by the glass being recorded. However, dust particles adhering to the outside surface of the capillaries, small fibres stemming from the agar used and glass scratches will also contribute to the background scatter, but the signals are small compared with those from colonies. Anyhow, care should be taken to clean the tubes before scanning and to use an agar as optically clean as possible. Nevertheless, such signal artifacts can never be completely avoided; suspect gel portions can easily be checked by personal stereomicroscopy. The possibility of following the growth of single colonies by daily scans is another useful feature to the capillary method [9]. Signal artifacts are easily detected since they do not “grow”. Daily scanning of single colonies, however, requires that the capillary tube remains in a fixed position since even slight positional changes of the tube considerably alter the peak height. This prerequisite is provided for in our capillary holder (fig. 2). Each peak, recorded as distribution of the intensity of the scattered light, is the product of the number of cells and their size or volume. It follows that a few cells of large volume may scatter as much light as many cells of small size, because the scanner cannot discriminate. However, since the amount of light scattered is directly proportional to the integrated cell volume a threshold may be set so that each colony of Exp CellRes 103(1976)
a minimum size of e.g. 50 pm is detected. Bowman et al. [12] and Abrams et al. [6] considered the number of pulses counted on day 0 as baseline, which was substracted from the daily counts. This procedure eliminates artificial pulses but requires an initial background scan and a second scan after incubation, yet well before other errors, such as colony coincidence and overgrowth emerge. However, such problems are rarely seen, since the number of colonies growing in one capillary is easily controlled by the number of hemopoietic cells seeded and are evenly distributed along the length of the gel [9]. While selecting the optimal scanning conditions among the various instruments settings, e.g. high voltage amplification, damping, scan and recorder speed, we found that there exists a definitive optimal light wavelength (fig. 2). This does not agree with previous claims by Zimmer & Neuhoff [8] that monochromacy is insignificant for light scattering measurements. Yet, an expensive monochromator could be replaced by a stabilized light source and a suitable filter with a fixed slit. The object of using the agar colony technique in capillary tubes was to test a large number of column fractions for their capacity to inhibit the multiplication of committed progenitor cells, i.e. possible chalone-like substances [3, 10, 151. Moreover, our automated method for cluster and colony counting of agar capillary tubes could also be of great value in evaluating colony-stimulating activities in a large number of samples. Such an endeavour required an assay, easy and quick to perform, yet offering satisfactory statistical reliability. This has been achieved by using enough self-contained test units (agar gels) and a scanning device together with a multiple capillary holder and an automatic
Automated scanning of bone marrow cell colonies sample changer, reducing personal manipulation considerably. In addition, we are now able to quantitatively follow the growth of individual colonies and thus determine any effects of growth regulators; this will reported elsewhere [9, lo]. We thank Mr H. Kohls for the skilful construction of the multiple capillary holder and the automatic capillarv changer. We are indebted to Dr 0. D. Laerum. Bergen (Norway), for continuing encouragement and Dr N. Iscove. Base1 (Switzerland) for valuable advice. This investigation was supported by the Deutsche Forschungsgemeinschaft (Ma 505/2 and 4).
REFERENCES 1. Pluznik. D H & Sachs, L. J cell camp physiol 66 (1965) 319. 2. Bradley, T R & Metcalf. D. Aust j exp biol med sci 44(1966) 287.
3. Muller-Berat, C N, Laerum. 0 D & Maurer, H R,
277
6th meet Eur study group cell prolif. hbstr.. p 4 1. Moscow (1973). 4. Maurer. H R, Weiss, G & Laerum, 0 D. Virchows Arch B cell path0120 (1976) 229. 5. Maurer, H R, Weiss, G & Laerum? 0 D, Blut. In press (1976). 6. Abrams, L, Carmeci, P, Bull, J M & Carbone, P P. J natl cancer inst 50 (1973) 267. 7. Maurer. H R & Henrv. R. Blut 33 (1976) II 8. Zimmer. H G & Ne&off, V. G-I-T Fachz Iab 19 (1975) 277. 9. Maurer, H R & Henry. R, Blut. In press (1976). 10. - In preparation (1976). Il. Metcalf. D, Control of proliferation in animal cells (ed B Clarkson & R B&erga‘l p. 887. Cold Spring Harbor Laboratory (1974). 12. Bowman, R L. Blume. P & Vurek, G G. Sckence 158 (1967) 78. 13. Braslow. NM &Bowman. R L, Science 175(1972) 1436. 14. Schoon. D J. Drake. J F, Fredrickson, A G, Tsuchiya, 4ppl Microbial 20 (1970) 815. 15. MacVittie, T J B McCarthy. K F, Exp hemarol 2 (1974) 182.
Received April 21, 1976 Accepted July 6. 1976
AUTOMATED SCANNING OF BONE MARROW CELL COLONIES GROWING IN AGAR-CONTAINING GLASS CAPILLARIES H. R. MAURER and R. HENRY Pharmazeutisches Institut der Freien Universitiit Berlin, D-1000 Berlin 33, Germany
SUMMARY An optical scanning system was developed to determine the growth of clusters and colonies of granulocytes and macrophages from mouse bone marrow cells in agar capillary tubes. The system consists of a commercially available photometer with a densitometer attachmen:, a two-mirror set to receive the light scattered by the cell colonies, a multiple capillary holder and an automatic sample changer. Parameters affecting scanning were examined and optimized: background scatter. instrument adjustments (e.g. signal damping) and threshold settings for clusters and colonies. Combined with the advantageous agar capillary technique, the complete scanning system provides an easy, accurate and sensitive method for rapid quantitation of hemopoietic cell colony formation in vitro.
The in vitro agar culture technique, originally introduced by Pluznik & Sachs [l] and Bradley & Metcalf [2], has had a large impact on present concepts of the proliferation and differentiation of hemopoietic progenitor cells. The visual examination of a great number of colonies as usually cultivated in Petri dishes, may become tedious and uneconomical when many Petri dishes have to be scored. This problem arose when we used the method to test for specific inhibitors (chalones) of granulopoiesis separated by biochemical techniques [3-51. We therefore adopted a modification of the agar colony method using glass capillary tubes [6] and studied in detail the parameters affecting the growth of mouse bone marrow cells in this system [7]. Its main advantage, amongst others, is that the colonies can be easily scanned on the basis of their light-scattering properties. However, the electronic counting, as performed by
Abrams et al. [6], requires photoelectric scanning equipment which is not commercially available. Upon our suggestion, Zimmer & Neuhoff [8] found a simple yet elegant way to adapt available photodensitometers to this purpose by means of a twomirror system. To scan ten capillary samples in sequence we have developed an automatic multiple sample changer that can be loaded with a multiple tube holder. This report describes the complete arrangement and the requirements for accurate and reproducible cluster and colony scanning. The various advantages offered by our automated technique will be demonstrated elsewhere 19, 101. MATERIALS
AND METHODS
Cell culture techniques The method of the in vitro agar culture of mouse bone marrow cells in glass capillary tubes of 1.38 mm internal diameter has been described in detail [?‘I. Meuse
272
Maurer and Henry Scattoreci
Pen recorder
Disc
light
dtmhment .?K
5
embryo conditioned medium was used as the source of colony stimulating activity in preference to mouse endotoxin-stimulated serum. Use of the former produced colonies of purely granulocytic cells whereas the latter gave colonies of highly vacuolized, large cells which were extremely difficult to identify. The preparation of mouse embryo conditioned medium is described elsewhere [9].
Scanning instruments We used a PMQ3 Photometer System with an automatic amplification unit, together with the ZK5 disc gel scanning attachment (Carl Zeiss, Oberkochen, Germany). The scanning attachment was adapted for light scattering measurements by use of two mirrors [8], and a built-in automatic sample changer enabled us to scan ten capillaries consecutively (fig. 1). The analog output of the amplifier unit was connected to a Servogor S, Type RE 541, linear potentiometric flat bed pen recorder (Goerz Electra GmbH, Vienna). The various settings of the scanning system, e.g. amplification slit width, damping, were adjusted to produce a pen deflection of about 90 7%when scanning the largest colonies grown in the agar gels, whilst retaining enough sensitivity to determine smaller cell clusters. We preferred the following settings for most scannings: PMQ3, high voltage amplification step 2, automatic amplification 10x, damping step 2, output voltage 10 mV, slit width 0.3 mm, wavelength 515 nm; ZKS, scan speed 30 mmlmin; Servogor S, recorder speed 60 mmlmin, sensitivity 20 mV.
Photometer PM0 3
system
so on. When the last, tenth capillary has been scanned, microswitch 2 reverses the direction of movement of the drive shaft and the holder returns to zero position. Microswitch 3 terminates the programme in zero position. The electronics controlling these movements are “grafted” onto the ZK5; thus this instruments can be still used for other scanning purposes. The time required to scan a capillary containing 100 pl of gel amounted to 3 min at a speed of 30 mm/mm of the ZK5, and the programme is completed in about 45 min.
RESULTS Fig. 1 shows the complete instruments system for scanning bone marrow cell colonies growing in agar contained in glass capillaries. Monochromatic light of optimal wavelength of 515 nm (fig. 2) is passed through the glass capillary moving through
6-
‘7
5-
/
When the programme is activated, the capillary holder with its ten glass capillary tubes moves from zero position in a horizontal direction through the light path thereby scanning the colonies in the first capillary. When the scan is complete a microswitch terminates the horizontal movement and induces a signal in the drive motor which lifts the capillary holder at 30 rpm 10 mm higher on a drive shaft. Microswitch 1 stops this movement and the second capillary is scanned and Exp
Cell
Res
103 (I 976)
/
‘A, ‘1.
4.
Mode of action of the automatic capillary changer (fig. 4)
Fig. 1. Scanning system for cell colonies growing in agar capillaries by light scattering. The system comprises a spectrophotometer (PMQ 3); a disc attachment (densitometer ZK 5) with built-in automatic capillary changer, an inserted multiple capillary holder and a twomirror set for light scattering detection; and a pen recorder. See Materials and Methods for details.
/
1.
\
./
1.
r
\.
4@J
4&J
MO
S&O
600
650
Fig. 2. Abscissa: light wavelength (nm); ordinate: peak height (cm). Scanning granulocytic colonies in agar capillaries at different wavelengths of the monochromatic light. Monochromator slit width 0.3 mm. For other conditions see Materials and Methods.
Automated scanning of bone marrow cell coiofzles
%\ ‘Rubber
gasket
I
5cm
,
Fig. 3. Multiple capillary holder (made from Plexiglasj. See Materials and Methods for details.
the light path of the disc gel attachment ZK 5. The capillary is held in a multiple capillary rack (fig. 3) carried by the automatic sample changer (fig. 4). The direct light path is blocked by the blackened back of mirror 2 fixed at an angle of 45” relative to the optical axis. Light scattered by the colonies in the capillary at an angle of 3045” is reflected by mirror 1 and received by the photomultiplier via mirror 2. Both mirrors of about 40 mm diameter are fixed onto a support plate and adjusted for maximum sensitivity. A slit width of 0.3 mm was chosen to give the best results, a reasonably flat baseline and a good signal response to colonies in the light path. A smaller slit width also produces a good baseline but only small peaks, whilst a larger slit produces a poor baseline and poor resolution. The slit height must exceed the diameter of the capillary and, as the light above and below the capillary will not be scattered, it does not contribute to the signal. A multple capillary rack was constructed to hold ten capillary tubes in a fixed, parallel and horizontal position (fig. 3). The holder itself consists of two frames, one
273
with four rivets and ten grooves for the capillaries, the other with four rubber gaskets to hold the tubes fast and four bores provided for the rivets to hold both frames together. The capillary holder can be easily put together and dismantled and is used for both the total period of incubation and the scanning procedure, obviating the need of further capillary manipulation. For automated scanning of ten capillaries in sequence, the holder with the fixed tubes is inserted into the automatic mulliple sample changer (fig. 4). This device moves the holder in meander-like path through t light beam. Its horizontal drive is provided by the disc attachment ZK5. while the vertical movement is given by a drive motor built into the sample changer. The resolving capacity of the scanning system is demonstrated by the photograph of several colonies (fig. 5). Evidently the peak height mainly depends on the cell concentration per unit volume, whereas t
4. Automatic multiple capillary changer. See Materials and Methods for details. The numbers I-10 designate the holder positions, (l)-(2) the microswitches.
Fig.
274
Maurer and Henry
20
0t-
capillary section; ordinate: scanner (pulse) count. Capillaries filled with water, 0.18 % agar in medium without and with lo4 mouse bone marrow cells/l00 ~1 were scanned under identical scanning conditions (see Materials and Methods).
Fig. 6. Abscissa:
Fig. 5. Granulocytic colonies in 0.18% agar medium
contained in a glass capillary tube (1.38 mm i.d.) with recorder scan, Range of cell no./colony: 235-296. Photo: B. Kamann.
peak area is the product of cell concentration and colony diameter. Background scatter mainly results from surface scratches on the glass tubes, electrostatically charged dust particles on the glass surface, particles contained in the agar and cell debris. Fig. 6 shows that the agar itself raises the background level and may produce small artifact peaks which, however, will not “grow” and can thus be distinguished from cell colonies. The sensitivity and resolution of the scanning system can be used in several ways depending upon the requirements of the operator. For example, from fig. 7, selecting damping step 6 provides the best Exp CeNRes 103 (1976)
signal-to-noise ratio and should be preferred. Other relevant instruments settings include high voltage amplification, scan speed, recorder speed and recorder sensitivity. For a quick evaluation of colony growth the capillaries are scanned at maximum possible speed (30 mmlmin) and all peaks above the pre-set threshold level are counted as colonies. Only when a colony grows directly behind another, i.e. along the optical axis, it is not possible to register a correct count. However, by regulating the
gel section; ordinate: pulse count. Selecting the optimum damping step (a-c): Signal/ noise ratio versus signal damping. Colonies at day 7 from lo4 bone marrow cells seeded per capillary. Scanning conditions see Materials and Methods.
Fig. 7. Abscissa:
Automated scanning of bone marrow cell colonies
Wlcq
CO”“t
no. of signals (pulses); ordinate: signal height (cm). Threshold settings for clusters and colonies. Clusters and colonies at day 7 from 10’ bone marrow cells seeded per capillary. Scanning conditions see Materials and Methods.
Fig. 8. Abscissa:
number of cells seeded this rarely occurs on statistical grounds. When a daily growth curve is desired the capillaries may be slowly scanned, producing larger peaks of greater area which may be accurately measured. Can the scanner discriminate between clusters and colonies? Clusters are, arbitrarily, defined as an accumulation of about 5-50 cells, whereas colonies consist of more than about 50 cells. The question of threshold setting was approached by the graph shown in fig. 8. Plotting the number of signals from a capillary versus the signal height yielded a curve with a decreasing slope relative to the abscissa; its inflection point was taken as cluster level. Visual counting using a stereomicroscope led to the colony level. Selecting a particular threshold level thus provided a fairly good accordance between eye and machine counting of clusters and especially, of colonies (fig. 9). DISCUSSION The principal advantages of culturing hemopoietic cells in agar contained in glass capillary tubes have been discussed at length
275
[6, 71. The cells forming clusters and colonies in agar have previously been identified as granulocytes, macrophages and mixtures of both, depending on the colonystimulating factor that was used [II]. Our studies have further documented the main feature of the capillary technique, i.e. its applicability to optical scanning. Previous reports [6, 12-14 on the use of light scattering for colony scanning have not led to widespread use, probably because suitable instruments were not available. BWetrH, the simple two-mirror system [g] can be adapted to many commerciai densitometers. The use of a multiple capillary holder and of an automatic sample changer has substantially improved the technique allowing us to accurately scan a great number of capillaries. All sera, media and samples containing colony-stimulating activities should be filtered through 0.2 pm membranes before use to produce as optically clear gels as possible. Bacterial colonies are much larger than the granulocytic coionies and are.
Fig. 9. Abscissa: visual colony counl (no. of colonies and clusters); ordinate: scanner count (no. of peaks).
0-O. Colonv and cluster count at 1 cm threshold height (r=O.982. szy= 3~1.82); Cm, colony count at 2 cm threshold height (r=0.995. s,,,=+O.71). Visual versus icanner counting of clusters and colonies at preset threshold levels of 1 and 7 cm. Clusters and-colonies at day 7 from 10’ bone marrow cells seeded per capillary. Scanning conditions see Materials and Methods.
276
Maurer and Henry
however, very seldom observed due to sterile operating procedures. Bacterial and fungal contamination during incubation is readily detected by cloudy gel ends which when scanned produce very large and broad signals. Therefore, provided that sterile, clean liquids are used, it can be seen (figs 6, 7) that the only signals produced above background scatter are, normally, those arising from cells and colonies inside the agar gel, no optical interference by the glass being recorded. However, dust particles adhering to the outside surface of the capillaries, small fibres stemming from the agar used and glass scratches will also contribute to the background scatter, but the signals are small compared with those from colonies. Anyhow, care should be taken to clean the tubes before scanning and to use an agar as optically clean as possible. Nevertheless, such signal artifacts can never be completely avoided; suspect gel portions can easily be checked by personal stereomicroscopy. The possibility of following the growth of single colonies by daily scans is another useful feature to the capillary method [9]. Signal artifacts are easily detected since they do not “grow”. Daily scanning of single colonies, however, requires that the capillary tube remains in a fixed position since even slight positional changes of the tube considerably alter the peak height. This prerequisite is provided for in our capillary holder (fig. 2). Each peak, recorded as distribution of the intensity of the scattered light, is the product of the number of cells and their size or volume. It follows that a few cells of large volume may scatter as much light as many cells of small size, because the scanner cannot discriminate. However, since the amount of light scattered is directly proportional to the integrated cell volume a threshold may be set so that each colony of Exp CellRes 103(1976)
a minimum size of e.g. 50 pm is detected. Bowman et al. [12] and Abrams et al. [6] considered the number of pulses counted on day 0 as baseline, which was substracted from the daily counts. This procedure eliminates artificial pulses but requires an initial background scan and a second scan after incubation, yet well before other errors, such as colony coincidence and overgrowth emerge. However, such problems are rarely seen, since the number of colonies growing in one capillary is easily controlled by the number of hemopoietic cells seeded and are evenly distributed along the length of the gel [9]. While selecting the optimal scanning conditions among the various instruments settings, e.g. high voltage amplification, damping, scan and recorder speed, we found that there exists a definitive optimal light wavelength (fig. 2). This does not agree with previous claims by Zimmer & Neuhoff [8] that monochromacy is insignificant for light scattering measurements. Yet, an expensive monochromator could be replaced by a stabilized light source and a suitable filter with a fixed slit. The object of using the agar colony technique in capillary tubes was to test a large number of column fractions for their capacity to inhibit the multiplication of committed progenitor cells, i.e. possible chalone-like substances [3, 10, 151. Moreover, our automated method for cluster and colony counting of agar capillary tubes could also be of great value in evaluating colony-stimulating activities in a large number of samples. Such an endeavour required an assay, easy and quick to perform, yet offering satisfactory statistical reliability. This has been achieved by using enough self-contained test units (agar gels) and a scanning device together with a multiple capillary holder and an automatic
Automated scanning of bone marrow cell colonies sample changer, reducing personal manipulation considerably. In addition, we are now able to quantitatively follow the growth of individual colonies and thus determine any effects of growth regulators; this will reported elsewhere [9, lo]. We thank Mr H. Kohls for the skilful construction of the multiple capillary holder and the automatic capillarv changer. We are indebted to Dr 0. D. Laerum. Bergen (Norway), for continuing encouragement and Dr N. Iscove. Base1 (Switzerland) for valuable advice. This investigation was supported by the Deutsche Forschungsgemeinschaft (Ma 505/2 and 4).
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Received April 21, 1976 Accepted July 6. 1976