Journal of Immunological Methods, 129 (1990) 23-30
23
Elsevier JIM 05539
A simple and rapid method to determine hematopoietic growth factor activity Vladimir K o t n i k * a n d W. R o b e r t F l e i s c h m a n n , Jr. Department of Microbiology, The Unioersity of Texas Medical Branch, Galveston, TX 77550, U.S.A.
(Received6 April 1989, revised received26 December 1989, accepted 27 December1989)
A rapid and simple colorimetric microassay method for the determination of hematopoietic growth factor activities was established. The assay was used to detect CSF-1, GM-CSF, and IL-3 activities. The assay was based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by metabolically active cells. Results obtained with the colorimetric microassay are comparable with those obtained with the soft agarose assay. Advantages of the colorimetric microassay include the conservation of reagents, the shorter incubation time for the experiment, the shorter assay time, and the ability to evaluate large numbers of samples. Key words: Hematopoieticgrowth factor; Bone marrow astray; Colorimetricassay; Tetrazolium salt
In~oduetion In 1966, Bradley and Metcalf developed a bone marrow colony formation assay in soft agar for the detection of hematopoietic growth factor activity. The assay is based on the ability of various progenitor cells to divide and from colonies of daughter cells in soft agar in the presence of specific growth factors. The number of colonies which develop has been shown to be directly correlated with the amount of growth factor present. This soft agar or agarose method is the standard method employed to study and define the properCorrespondence to." W.R. Fleischrnarm, Jr., Department of Microbiology, The University of Texas Medical Branch, Galveston, TX 77550, U.S.A. * Current address: Medical Faculty, Institute of Microbiology, 61105 Ljubljana, Yugoslavia. Abbreviations: CSF-1, rnacrophage colony stimulating factor; rGM-CSF, recombinant DNA-derivedgranuloeyte-rnacrophage colony stimulating factor; I1--3, interleukin-3; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
ties of different hematopoietic growth factors (Metcalf, 1984). For some purposes, it is indispensable. However, it is a time-consuming and laborious assay to perform. It also requires substantial quantities of reagents. A more rapid and less time-consuming assay which also uses smaller quantities of reagents is needed for routine assays. To address this need, we have developed a colorimetric assay using bone marrow cells grown in microtest plates. This assay is a modification of the colorimetric assay developed by Tada et al. (1986) for measuring interleukin-2 activity and by K.riegler et al. (1987) for measuring macrophage growth factors. The colorimetric method quantities the proliferation of bone marrow, cells by the increase in their mitochondrial activity. The mitochondrial activity is determined by the reduction of the tetrazolium salt 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into crystaline formazan (Mosmann, 1983). After dissolving the blue formazan crystals, the absorbance of the solution can be
0022-1759/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
24
read to 590 nm. The absorbance directly correlates with the concentration of the growth factor used.
Materials and methods
Mice Female C57BL/6 mice purchased from TimCo Breeding (Houston, TX) were used as the source of bone marrow cells for the hematopoietic colony assays.
Hematopoietic growth factors Recombinant DNA-derived murine GM-CSF (granulocyte/macrophage colony stimulating factor) and natural murine IL-3 (multipotential colony stimulating factor) were purchased from Genzyme (Boston, MA). Natural murine CSF-1 (macrophage colony stimulating factor) was produced in mouse L-929 fibroblast cells and was purified from serum-free Eagle's MEM conditioned medium (Gibco, Grand Island, NY) according to the method of Waheed and Shadduck (1979).
Preparation of bone marrow cells Nucleated bone marrow cells were extracted from female C57BL/6 mice by skinning and amputating the rear legs of mice killed by cervical dislocation; detaching the muscles from the femurs; and, flushing the marrow from the femurs with Alpha MEM (Gibco, Grand Island, NY) using a 23 gauge needle inserted in the lumen of the bone. The bone marrow cells were gently dissociated and washed by centrifugation. Red blood cells were lysed by treatment at 4 °C for 2 rain with 0.83% NH4CI. The cells were washed again by centrifugation, resuspended in a minimal volume of RPMI (Gibco) supplemented with 10% donor calf serum, and counted in a hemacytometer.
Soft agarose bone marrow assay for hematopoietic growth factors The soft agarose bone marrow assay was performed in 35 mm plastic gridded tissue culture dishes (2 mm grid, LUX, Miles, Naperville, IL) as a modification of the double-layer agar technique originally described by Bradley and Metcalf
(1966). The bone marrow cells were prepared as described and were resuspended at a concentration of 1.5 × 105 cells/ml in warm (40°C) top agarose which was prepared by supplementing Alpha MEM with 0.35% agarose, 15% donor calf serum (Hazelton Dutchland, Denver, PA), 2.5 x 10-5 M 2-mercaptoethanol, and antibiotics (penicillin, 100 U/ml, Pfizer, New York, NY; streptomycin, 100/~g/ml, Pfizer; and gentamycin, 11 #g/ml, Invenex, Chagrin Falls, OH). 500 #1 aliquots of the bone marrow cell suspension in top agarose were layered over 1.5 ml of solidified bottom agarose containing Alpha MEM supplemented with 0.70% agarose, 15% donor calf serum, 2.5 x 10 -5 M 2-mercaptoethanol, and antibiotics in 35 × 10 mm gridded tissue culture dishes. After the top agarose solidified upon cooling, 0.5 ml of different concentrations of hematopoietic growth factors was added and the dishes were incubated at 37 °C for 7 days in a well-humidified 7.5% CO2 atmosphere. Colonies containing 50 or more cells were scored using a Nikon inverted microscope at 40 × magnification. All experiments were performed in triplicate and at least four two-fold serial dilutions were made for each sample.
Colorimetric bone marrow hematopoietic growth factors
microassay for
The colorimetric bone marrow microassay was performed in flat bottom 96-well tissue culture microtest plates (Falcon) as a modification of the colorimetric assay previously described by Mosmann (1983) and Tada et al. (1986). Serial two-fold dilutions of the growth factors were made by serially diluting 50 /.tl aliquots of growth factor into 50 ttl aliquots of Alpha MEM supplemented with 30% donor calf serum, 2.5 x 10 -s M 2-mercaptoethanol, and antibiotics which had been previously added to the wells of the microtest plates. Then, the bone marrow cells were prepared as described and were resuspended at 1.5 x 10 6 cells/ml in Alpha MEM supplemented with 30% d o n o r calf s e r u m , 2.5 x 10- ~ M 2mercaptoethanol, and antibiotics. 50/~1 aliquots of the bone marrow cell suspension were added to the growth factor dilutions in each well in the microtest plates. The microtest plates were incubated at 37°C for 3 days in a well-humidified
25 7.5% CO2 atmosphere. This represented an incubation period which was four days shorter than that used in the soft agarose bone marrow assay. The colorimetric microassay was performed as follows. The indicator solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium b r o m i d e (MT'F, Sigma, St. Louis, MO), was prepared as a 5 m g / m l solution in phosphate-buffered saline and stored for 1-1.5 months in a light protected container at 4 ° C . After 3 days of incubation of the bone marrow cells, 20/~1 of the MTT was added to each of the wells of the microtest plates. The cells were incubated for another 4 - 5 h at 3 7 ° C . During this time, the MTT was reduced by rnitochondrial enzymes in the bone marrow cells to blue crystals of formazan. The formazan crystals were then dissolved by adding 100 /xl of a 10% solution of sodium dodecyl sulfate (Sigma) in 0.01 N HC1 and by further incubating the samples at 3 7 ° C for 4 - 2 0 h protected from light. The formazan concentrations were quantitated using an EIA reader (590 nm wavelength filter; Bio-Rad Laboratories, Richmond, CA) connected through a Bio-Rad software program (MacReader) to a Macintosh computer (Apple Computer, Cupertino, CA). Blank control wells consisting of bone marrow cells incubated with growth medium but without hematopoietic growth factors were included for each assay. Absorption values for the blank controls were automatically subtracted from the absorption values for the test wells. All experiments were performed in quadruplicate.
Results
Effect of cell concentration on detection of bone marrow cell growth in the soft agarose assay and in the colorimetric microassay A colorimetric bone marrow microassay was developed using bone marrow cells grown in liquid medium in the wells of microtest plates. It was necessary to determine whether the colorimetric bone marrow microassay would give results which would be reproducible and comparable to the results obtained with a soft agarose bone marrow assay. Cell concentrations from 7.5 x 10 4 to 2.4 × 10 6 cells/well were examined in the colorimetric microassay. 3 days after plating, the cells were
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Fig. ]. Comparisons of soft agarosc assay and colorimetric microassay using different concentrations of bone marrow
cells. Bone marrow cells were grown at cell concentration from 7.5 x 104 to 2.4 × 106 cells/plate in soft agarose assay ([3) and at cell concentrations from 7.5 × 104 to 2.4 x 106 cells/well in the colorimetric microassay (11). A single concentration of 16 U/ml of natural murine CSF-1 was used in both assays to stimulate bone marrow cell proliferation. For the soft agarose assay, cells were grown in soft agarose on 35 mm petri plates for 7 days before colonies were counted using a Nikon microscope (40 X magnification). The experiment was perfon-ned in triplicate. For the coiorimetric microassay,cells were grown on rnicrotest plates for 3 days before the ability to metabolize 3-(4,5-dimetliylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (M'I'F) was determined. 20/xl of the MTT was added to each of the wells of the microtest plates. The cells were incubated for 4 h at 37 ° C. D.ufing this time, the MTT was reduced by mitoebondrial enzymesin the bone marrowcells to blue crystals of formazan. The formazan crystals were then dissolved by adding 100/~! of a 10% solution of sodium dodecyl sulfate in 0.01 N HCI and by further incubating the samples at 37o C for 16 h. The experiment was performed in quadruplicate. Points, means; bars, standard errors. examined for their ability to reduce M ' I T to formazan d y e and for the intensity of absorption which they produced. For purposes of comparison, the same range of bone marrow cell concentrations was examined in a soft agarose assay for their ability to form colonies. The data of a representative experiment are shown in Fig. 1. In agreement with previous studies (see Metcalf, 1984), the data show that the ability to form colonies in soft agarose was related in a dose-dependent fashion to the concentration of bone marrow ceils employed. The data also show that the reduction of M T T and thus the intensity of absorption was related in a similar dose-dependent fashion to the concentration of bone marrow cells employed. Thus, the soft agarose assay and the colorimetric microassay gave similar results.
26
Colorimetric assays are most accurate when the intensity of absorption is between 0.2 and 1 . 2 0 D . The intensities of absorption reported in Fig. 1 ranged from approximately 0.05 to 0.27 OD. However, it should be noted that these reported intensities are corrected values. The background intensities of absorption of wells in the microtest plates containing control cells plated in the absence of CSF-1 were always about 0 . 4 0 D and were automatically subtracted from the intensities of absorption in the test wells by our computer program. For example, for the range of cell concentrations expressed on Fig. 1 (7.5 × 104-2.4 × 106 cells/well), the intensi ties of absorption ranged from approximately 0.4 to 0.7. Thus, the values reported for the colorimetric microassay in Fig. 1 and in all subsequent figures lie within the appropriate absorption range (0.2-1.20D). Effect of CSF-I concentration on detection of bone marrow cell growth in the soft agarose assay and in the colorimetric microassay The growth of bone marrow cells in a soft agarose assay is proportional to the amount of growth factor to which the bone marrow cells are exposed. It was necessary to determine whether the colorimetric microassay would given comparable results. Bone marrow cells were grown either in the soft agarose assay or in the colorimetric microassay in the presence of concentrations of CSF-1 .ranging from 6.25 to 100 U/ml. The averaged data of four experiments are presented in Fig. 2. The data show that the growth of cells in the soft agarose assay is proportional to the concentration of CSF-1 employed. The growth of colonies in the colorimetric microassay was similarly proportional to the concentration of CSF-1 employed. Thus, the soft agarose assay and the colorimetric microassay gave comparable results. Calculation of CSF-! units in the colorimetric microassay A method of calculating CSF-1 activity has been established for the soft agarose assay (Koren et al., 1986). The method is based on the calculation of the linear regression line through the "linear' (actually log-linear) part of the titration curve. The titer of CSF-I is defined as the reciprocal of the dilution of CSF-1 required to give one bone
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Fig. 2. Comparisons of soft agarose assay and colorimetric microassay using different concentrations of CSF-1. Bone marrow cells were grown as described in Fig. I at a concentration of 7.5 x 104 cells/plate for the soft agarose assay (13) and at a concentration of 7.5 × 104 cells/well in the colorimetric microassay (n) in the presence of concentrations of CSF-I ranging from 6.25 to 100 U / m l . Data for the soft agarose assays are expressed as the aritl-,,netic m e a n s of four experiments performed in triplicate. Data for the colorimetric assays are expressed as the arithmetic m e a n s of four experiments performed in quadruplicate. Points, means; bars, standard errors.
marrow colony (extrapolated from the linear regression line). Such a definition of titer is easily applied to the colorimetric microassay. Fig. 3 presents a representative example of a titration of the laboratory standard CSF-1 preparation and of an unknown CSF-1 preparation Linear regression lines can be determined for the two CSF-1 preparations. By extrapolating each ol the linear regression lines to the abscissa, thc CSF-1 titer can be expressed as the reciprocal o! the dilution of CSF-1 which gives 0.00 absorbance. In this example, the laboratory standard has a titer of 752 U / m l in the colorimetric microassay. The unknown preparation has a titer of 34~ U / m l in the colorimetric microassay. Since th~ laboratory standard preparation has a known titeJ of 640 U / m l with the soft agarose assay, a simpk proportion can be set up and the titer of th~ unknown preparation can be predicted to be 29.U / m l . The actual titer on soft agarose of th( unknown preparation was determined to be 32(. U/trd, a difference of 11% and well within tht standard error of the assay. Alternatively, sinc~ the slopes for all samples for a given cytokine art essentially identical in a given assay, a GRIDL5
27
Effect of time of M T T reduction on the colorimetric microassay
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Fig. 3. Titration of a CSF-I preparation of unknown titer. Bone marrow cells at a cell concentration of 7.5 x 104 cells/well were grown as described in Fig. 1 for the colorimetric microassay in the presence of various dilutions of the laboratory standard CSF-1 preparation of known titer (121) or in the presence of a CSF-1 preparation of unknown titer (11). The data are expressed as the arithmetic means of a representative experiment performed in qaudruplicate. Points, means; bars, standard errors•
T h e c o l o r i m e t r i c a s s a y is d e p e n d e n t on the r e d u c t i o n o f M T T b y m i t o c h o n d r i a l enzymes to f o r m f o r m a z a n c r y s t a l s in the cells. In o r d e r to m e a s u r e the a m o u n t o f f o r m a z a n , the crystals m u s t b e d i s s o l v e d ( b y a d d i n g a s o l u t i o n of 10% s o d i u m d o d e c y l sulfate in 0.01 N HCI ( S D S / H C 1 solution)) b e f o r e the a m o u n t of f o r m a z a n can be e s t i m a t e d in an E I A reader. T w o c o m p e t i n g factors are involved in d e t e r m i n g the most app r o p r i a t e t i m e to r e a d the i n t e n s i t y of the formaTan: first, it takes s o m e time to dissolve the f o r m a z a n crystals a n d second, the intensity o f the f o r m a z a n d y e d e c r e a s e s with time after t r e a t m e n t with S D S / H C 1 . Fig. 4 p r e s e n t s the results of a n e x p e r i m e n t which c o m p a r e s the loss o f intensity of formazen with i n c r e a s i n g time after S D S / H C I treatment. T h e d a t a s h o w that the f o r m a z a n was most intense in its color 4 hours after t r e a t m e n t with SDS~-IC1. H o w e v e r , m i c r o s c o p i c e x a m i n a t i o n of the microtest p l a t e wells i n d i c a t e d that there were some
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(grid search least squares fit for a n o n l i n e a r function) (Bevington, 1969) p r o g r a m can b e used to d e t e r m i n e the titer. U s i n g the f o r m u l a y = a (log x ) + b, the x i n t e r c e p t can b e d e t e r m i n e d as x i n t e r c e p t = 10 -(b/°). Since the titer is the reciprocal of the x intercept, titer = 10 (b/a). U s i n g the G R I D L S p r o g r a m , the titers of the l a b o r a t o r y s t a n d a r d p r e p a r a t i o n a n d of the u n k n o w n prep a r a t i o n c a n b e d e t e r m i n e d to b e 672 U / m l a n d 340 U / m l , respectively, in the c o l o r i m e t r i c assay. By simple p r o p o r t i o n a l i t y , the u n k n o w n p r e p a r a tion can b e p r e d i c t e d to have a titer o f 324 U / m l o n soft agarose. Since the a c t u a l titer o f the unk n o w n p r e p a r a t i o n o n soft a g a r o s e is 329 U / m l , the difference o f 1.5% is well w i t h i n the s t a n d a r d e r r o r of the assay. Thus, if a l a b o r a t o r y s t a n d a r d of k n o w n titer with the soft a g a r o s e assay is i n c l u d e d as a reference p r e p a r a t i o n , the relative titers can b e det e r m i n e d b y the c o l o r i m e t r i c m i c r o a s s a y a n d these titers can b e n o r m a l i z e d to the soft a g a r o s e titer of the l a b o r a t o r y s t a n d a r d p r e p a r a t i o n to give an e s t i m a t e d soft a g a r o s e titer for a n u n k n o w n C S F - 1 preparation.
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Fig. 4. Effect of time of MTT reduction on the colorimetric microassay. Bone marrow cells at a cell concentration of 7.5 x 10 4 cells/well were grown as described in Fig. 1 for the colorimetric microassay. After MTT was added to the cells, the cells were incubated for 4 h during which time the MTT was reduced by mitochondrial enzymes in the bone marrow cells to blue crystals of formazan. The formazan crystals were then dissolved by adding 100 pl of a 10% solution of sodium dodecyl sulfate in 0.01 N HC1. The samples were then incubated at 37°C for 4 h ( t ) , 16 h (11), or 40 h (A). The data are expressed as the arithmetic means of a representative experiments performed in quadruplicate. Points, means; bars. standard errors.
28 undissolved crystals present. The formazan was less intense in its color 16 h after treatment with SDS/HC1, a time when the formazan crystals were all dissolved. The curve generated 40 h after S D S / H C I addition showed that the intensity of the formazan continued to decrease with additional time after treatment with S D S / H C I . It should be noted that the titers of the CSF-1 (determined by extrapolation of the linear regression lines) were essentially identical for the three times of treatment with S D S / H C I (data not shown). Thus, although the intensity of the formazan dye decreased with increasing time of exposure of the formazan to SDS/HC1, the time of treatment with SDS/HC1 did not affect the determination of CSF-1 titer.
Measurement of r G M - C S F and 1L-3 actwities in the soft agarose assay and in the colorimetric microassay CSF-1 predominantly stimulates the gro ~¢th of macrophages. To test the general applicabiiity of the colorimetric microassay for detection of hematopoietic growth factors other than CSF-1, the soft agarose assay and the colorimetric microassay were evaluated for their abilities to detect
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Fig. 5. Comparisons of soft agarose assay and colorimetric microassay using different concentrations of rGM-CSF. Bone marrow cells were grown as described in Fig. 1 at a concentration of 7.5 × 104 cells/plate for the soft agarose assay (D) and at a concentration of 7.5 × 104 cells/well in the colorimetric microassay (I) in the presence of concentrations of rGM-CSF ranging from 3.1 to 100 U/ml. Data for the soft agarose assays are expressed as the arithmetic means of five experiments performed in triplicate. Data for the colorimetric assays are expressed as the arithmetic means of five experiments performed in quadruplicate. Points, means; bars, standard errors.
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Fig. 6. Comparisons of soft agarose assay and colorimetric microassay using different concentrations of IL-3. Bone marrow cells were grown as described in Fig. 1 at a cell concentration of 7.5 × 104 cells/plate for both the soft agarose assay (El) and at a concentration of 7.5 × 104 cells/well in the colorimetric microassay (11) in the presence of concentrations of IL-3 ranging from 3.1 to 100 U/ml. Data for the soft agarose assays are expressed as the arithmetic means of two experiments performed in quadruplicate. Points, means; bars. standard errors. G M - C S F and IL-3. GM-CSF stimulates the growth of granulocytes and macrophages while IL-3 stimulates the growth of erythroid progenitor cells, multipotential progenitor cells, eosinophils, granulocytes, macrophages, and lymphocytes (for review see Metcalf, 1984). Bone marrow cells were grown either in the soft agarose assay or in the colorimetric microassay in the presence of rGMCSF or IL-3. Fig. 5 presents the averaged data of five experiments using rGM-CSF. The data show that both the growth of colonies in the soft agarose assay and the growth of cells in the colorimetric microassay were similarly proportional to the concentration of r G M - C S F employed. Fig. 6 presents the averaged data of two experiments using IL-3. Again the data show that both the growth of colonies in the soft agaros6 assay and the growth of cells in the colorimetric microassay were similarly proportional to the concentration of IL-3 employed. Thus, the colorimetric microassay has a general a p p l i c a t i o n for the detection of hematopoietic growth factors.
Discussion
A colorimetric rnicroassay was developed by Mosmann (1983) which was based upon the
29 metabolism of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyhetrazolium bromide (MTT) to a blue formazan metabolite. The colorimetric microassay employed microtest plates and an ELISA Reader to measure IL-2- and mitogeninduced proliferation. Tada et al. (1986) further modified this microassay for measuring IL-2 activity. This report presents an adaptation of Tada et al.'s microassay method for the detection and quantitation of hematopoietic growth factors. The colorimetric microassay reported here employs 96-well microtest plates and permits the assay of many samples with minimal effort. The assays can be read directly in the wells of the microtest plates in a EIA Reader without transferring the samples. Using a personal computer and appropriate software (in this case a Macintosh computer and MacReader software), the data can be evaluated quickly. The assay has been shown to be useful for the detection and quantitation of a variety of hematopoietic growth factors including CSF-1, GM-CSF, and IL-3. The response of the bone marrow cells to these hematopoietic growth factors can be seen with the colorimetric microassay to be proportional to the amount of growth factor added over a wide range of growth factor concentrations. The data obtained with the colorimetric microassay are very similar to those generated with the soft agar assay of Bradley and Metcalf (1966). The colorimetric microassay permits the determination of the titers of hematopoietic growth factors and, by using a laboratory standard, one can normalize these titers to those generated by the soft agar assay. It should be noted while the formazan dye decays with time after treatment with 10% SDS in 0.01 N HC1, the titers determined by the colorimetric microassay are independent of the decay of the formazan dye. The colorimetric microassay has a number of advantages over the soft agar assay of Bradley and Metcalf (1966) for the detection of hematopoietic factor activity. First, the colorimetric microassay requires the expenditure of relatively smaller quantities of reagents which are often difficult to obtain. The soft agar assay is performed in 35 mm plastic petri dishes and, as such, it requires the expenditure of 2.5 ml of reagents. The colorimetric
microassay uses only 0.1 ml of reagents. Second, the colorimetric microassay is completed in a shorter period of time. The soft agar assay is dependent on the development of clones of cells into colonies and, as such, it requires an incubation period of 7-14 days. The colorimetric microassay can be read following a 3-day incubation period. Third, the colorimetric microassay is relatively less time consuming to evaluate. The soft agar assay is dependent on the microscopic examination of the plates to identify colonies as clones of 50 or more cells or as clones of cells with diameters of > 0.5 mm (Kriegler et al., 1987) and, as such, is time consuming, labor intensive, and somewhat subjective. The colorimetric microassay can be completed in minutes rather than hours. Finally, the colorimetric microassay can be used for the assay of large numbers of samples. One limitation of the colorimetric assay is that it cannot be used to distinguish between colonies and clusters. Another is that the colorimetric,-assay can not be used to type the colonies which have been stimulated to grow (i.e., macrophage colonies, granulocyte colonies, etc.). Recently, Kriegler et al. (1987) have developed a colorimetric tetrazolium assay to measure the activity of hematopoietic growth factors which stimulate the growth of macrophage progenitor cells. Their assay employed six-well cluster dishes (35 mm diameter) which required the investment of relatively large quantities of reagents. The method also required the manipulation of the samples before they could be read individually in a spectrophotometer. The colorimetric microassay reported here has several of the same advantages over the colorimetric assay of Kriegler et al. (1987) as it has over the soft agar assay of Bradley and Metcalf (1966): the colorimetric microassay uses smaller quantities of reagents; the colorimetric microassays can be read very quickly; and, the colorimetric microassay permits the assay of large number of samples in the same experiment. It should be noted that the soft agar or agarose assay has become the standard procedure for the determination of hematopoietic growth factor concentration and activity. Using the soft agar assay method, the activity of growth factors can be expressed in units. 1 U is defined as the amount of growth factor which must be present for the
30 development of one colony, as determined by extrapolating the linear regression line drawn through the linear portion of the growth factor titration curve. The colorimetric microassay gives a titration curve similar to that observed with the soft agar assay. Thus, the colorimetric microassay also permits an expression of growth factor titer in units by the extrapolation of its linear regression line. Using these definitions of units, the colorimetric microassay detects fewer units than the soft agar assay. For example, the laboratory standard has a titer of 188 U with the colorimetric microassay and 640 U with the soft agar assay. However, if a l a b o r a t o r y s t a n d a r d p r e p a r a t i o n of hematopoietic growth factor of known titer in the soft agar assay is included with each colorimetric microassay, the titers of each unknown can be normalized to the laboratory standard preparation and expressed according tn their relative expected titers in the soft agar assay. In summary, the colorimetric microassay can be used instead of the more cumbersome soft agar assay for a variety of experimental purposes. It is not anticipated that it could completely replace the soft agar assay. Because the colorimetric microassay does not examine clones microscopically, it does not permit the differentiation of different types of colonies (i.e., macrophage, granulocyte) which the soft agar assay permits.
Acknowledgements We thank Antonija Kotnik for her excellent technical assistance and William Law for his statistical assistance.
This study was supported by USPHS Grant CA26475 awarded by the National Cancer Institute, Department of Health and Human Services and by Grant IFN-3 awarded by the American Cancer Society.
References Bevington, P.R. (1969) Data Reduction and Error Analysis for the Physical Sciences. McGraw-Hill, New York. Bradley, T.R. and Metcalf, D. (1966) The growth of mouse bone marrow cells in vitro. Aust. J. Exp. Biol. Med. Sci. 44, 287. Koren, S., Klimpel, G.R. and Fleischmann, Jr., W.R. (1986) Treatment of mice with macrophage colony stimulating factor (CSF-I) prevents in vivo myelosuppression induced by murine alpha, beta and gamma interferons. J. Biol. Resp. Modif. 5, 481. Kriegter, A.B., Bradley, T.R., Hodgson, G.S. and McNiece, I.K. (1987) A colorimetric liquid culture assay of a growth factor for primitive murine macrophage progenitor cells. J. Immunol. Methods 103, 93. Metcalf, D. (1984) The Hemopoietic Colony Stimulating Factors. Elsevier, Amsterdam, p. 55. Mosmann, T. (1983) Rapid colonmetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. lmmunol. Methods 65, 55. Tada, H., Shiho, O, Kurushima, K., Koyama, M. and Tsukamoto, K. (1986) An improved colorimetric assay for interleukin 2. J. lmmunol. Methods 93, 157. Waheed, A. and Shadduck, R.K. (1979) Purification and properties of L cell-derived colony-stimulating factor. J. Lab. Clin. Med. 94, 180.
23
Elsevier JIM 05539
A simple and rapid method to determine hematopoietic growth factor activity Vladimir K o t n i k * a n d W. R o b e r t F l e i s c h m a n n , Jr. Department of Microbiology, The Unioersity of Texas Medical Branch, Galveston, TX 77550, U.S.A.
(Received6 April 1989, revised received26 December 1989, accepted 27 December1989)
A rapid and simple colorimetric microassay method for the determination of hematopoietic growth factor activities was established. The assay was used to detect CSF-1, GM-CSF, and IL-3 activities. The assay was based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by metabolically active cells. Results obtained with the colorimetric microassay are comparable with those obtained with the soft agarose assay. Advantages of the colorimetric microassay include the conservation of reagents, the shorter incubation time for the experiment, the shorter assay time, and the ability to evaluate large numbers of samples. Key words: Hematopoieticgrowth factor; Bone marrow astray; Colorimetricassay; Tetrazolium salt
In~oduetion In 1966, Bradley and Metcalf developed a bone marrow colony formation assay in soft agar for the detection of hematopoietic growth factor activity. The assay is based on the ability of various progenitor cells to divide and from colonies of daughter cells in soft agar in the presence of specific growth factors. The number of colonies which develop has been shown to be directly correlated with the amount of growth factor present. This soft agar or agarose method is the standard method employed to study and define the properCorrespondence to." W.R. Fleischrnarm, Jr., Department of Microbiology, The University of Texas Medical Branch, Galveston, TX 77550, U.S.A. * Current address: Medical Faculty, Institute of Microbiology, 61105 Ljubljana, Yugoslavia. Abbreviations: CSF-1, rnacrophage colony stimulating factor; rGM-CSF, recombinant DNA-derivedgranuloeyte-rnacrophage colony stimulating factor; I1--3, interleukin-3; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
ties of different hematopoietic growth factors (Metcalf, 1984). For some purposes, it is indispensable. However, it is a time-consuming and laborious assay to perform. It also requires substantial quantities of reagents. A more rapid and less time-consuming assay which also uses smaller quantities of reagents is needed for routine assays. To address this need, we have developed a colorimetric assay using bone marrow cells grown in microtest plates. This assay is a modification of the colorimetric assay developed by Tada et al. (1986) for measuring interleukin-2 activity and by K.riegler et al. (1987) for measuring macrophage growth factors. The colorimetric method quantities the proliferation of bone marrow, cells by the increase in their mitochondrial activity. The mitochondrial activity is determined by the reduction of the tetrazolium salt 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into crystaline formazan (Mosmann, 1983). After dissolving the blue formazan crystals, the absorbance of the solution can be
0022-1759/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
24
read to 590 nm. The absorbance directly correlates with the concentration of the growth factor used.
Materials and methods
Mice Female C57BL/6 mice purchased from TimCo Breeding (Houston, TX) were used as the source of bone marrow cells for the hematopoietic colony assays.
Hematopoietic growth factors Recombinant DNA-derived murine GM-CSF (granulocyte/macrophage colony stimulating factor) and natural murine IL-3 (multipotential colony stimulating factor) were purchased from Genzyme (Boston, MA). Natural murine CSF-1 (macrophage colony stimulating factor) was produced in mouse L-929 fibroblast cells and was purified from serum-free Eagle's MEM conditioned medium (Gibco, Grand Island, NY) according to the method of Waheed and Shadduck (1979).
Preparation of bone marrow cells Nucleated bone marrow cells were extracted from female C57BL/6 mice by skinning and amputating the rear legs of mice killed by cervical dislocation; detaching the muscles from the femurs; and, flushing the marrow from the femurs with Alpha MEM (Gibco, Grand Island, NY) using a 23 gauge needle inserted in the lumen of the bone. The bone marrow cells were gently dissociated and washed by centrifugation. Red blood cells were lysed by treatment at 4 °C for 2 rain with 0.83% NH4CI. The cells were washed again by centrifugation, resuspended in a minimal volume of RPMI (Gibco) supplemented with 10% donor calf serum, and counted in a hemacytometer.
Soft agarose bone marrow assay for hematopoietic growth factors The soft agarose bone marrow assay was performed in 35 mm plastic gridded tissue culture dishes (2 mm grid, LUX, Miles, Naperville, IL) as a modification of the double-layer agar technique originally described by Bradley and Metcalf
(1966). The bone marrow cells were prepared as described and were resuspended at a concentration of 1.5 × 105 cells/ml in warm (40°C) top agarose which was prepared by supplementing Alpha MEM with 0.35% agarose, 15% donor calf serum (Hazelton Dutchland, Denver, PA), 2.5 x 10-5 M 2-mercaptoethanol, and antibiotics (penicillin, 100 U/ml, Pfizer, New York, NY; streptomycin, 100/~g/ml, Pfizer; and gentamycin, 11 #g/ml, Invenex, Chagrin Falls, OH). 500 #1 aliquots of the bone marrow cell suspension in top agarose were layered over 1.5 ml of solidified bottom agarose containing Alpha MEM supplemented with 0.70% agarose, 15% donor calf serum, 2.5 x 10 -5 M 2-mercaptoethanol, and antibiotics in 35 × 10 mm gridded tissue culture dishes. After the top agarose solidified upon cooling, 0.5 ml of different concentrations of hematopoietic growth factors was added and the dishes were incubated at 37 °C for 7 days in a well-humidified 7.5% CO2 atmosphere. Colonies containing 50 or more cells were scored using a Nikon inverted microscope at 40 × magnification. All experiments were performed in triplicate and at least four two-fold serial dilutions were made for each sample.
Colorimetric bone marrow hematopoietic growth factors
microassay for
The colorimetric bone marrow microassay was performed in flat bottom 96-well tissue culture microtest plates (Falcon) as a modification of the colorimetric assay previously described by Mosmann (1983) and Tada et al. (1986). Serial two-fold dilutions of the growth factors were made by serially diluting 50 /.tl aliquots of growth factor into 50 ttl aliquots of Alpha MEM supplemented with 30% donor calf serum, 2.5 x 10 -s M 2-mercaptoethanol, and antibiotics which had been previously added to the wells of the microtest plates. Then, the bone marrow cells were prepared as described and were resuspended at 1.5 x 10 6 cells/ml in Alpha MEM supplemented with 30% d o n o r calf s e r u m , 2.5 x 10- ~ M 2mercaptoethanol, and antibiotics. 50/~1 aliquots of the bone marrow cell suspension were added to the growth factor dilutions in each well in the microtest plates. The microtest plates were incubated at 37°C for 3 days in a well-humidified
25 7.5% CO2 atmosphere. This represented an incubation period which was four days shorter than that used in the soft agarose bone marrow assay. The colorimetric microassay was performed as follows. The indicator solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium b r o m i d e (MT'F, Sigma, St. Louis, MO), was prepared as a 5 m g / m l solution in phosphate-buffered saline and stored for 1-1.5 months in a light protected container at 4 ° C . After 3 days of incubation of the bone marrow cells, 20/~1 of the MTT was added to each of the wells of the microtest plates. The cells were incubated for another 4 - 5 h at 3 7 ° C . During this time, the MTT was reduced by rnitochondrial enzymes in the bone marrow cells to blue crystals of formazan. The formazan crystals were then dissolved by adding 100 /xl of a 10% solution of sodium dodecyl sulfate (Sigma) in 0.01 N HC1 and by further incubating the samples at 3 7 ° C for 4 - 2 0 h protected from light. The formazan concentrations were quantitated using an EIA reader (590 nm wavelength filter; Bio-Rad Laboratories, Richmond, CA) connected through a Bio-Rad software program (MacReader) to a Macintosh computer (Apple Computer, Cupertino, CA). Blank control wells consisting of bone marrow cells incubated with growth medium but without hematopoietic growth factors were included for each assay. Absorption values for the blank controls were automatically subtracted from the absorption values for the test wells. All experiments were performed in quadruplicate.
Results
Effect of cell concentration on detection of bone marrow cell growth in the soft agarose assay and in the colorimetric microassay A colorimetric bone marrow microassay was developed using bone marrow cells grown in liquid medium in the wells of microtest plates. It was necessary to determine whether the colorimetric bone marrow microassay would give results which would be reproducible and comparable to the results obtained with a soft agarose bone marrow assay. Cell concentrations from 7.5 x 10 4 to 2.4 × 10 6 cells/well were examined in the colorimetric microassay. 3 days after plating, the cells were
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Fig. ]. Comparisons of soft agarosc assay and colorimetric microassay using different concentrations of bone marrow
cells. Bone marrow cells were grown at cell concentration from 7.5 x 104 to 2.4 × 106 cells/plate in soft agarose assay ([3) and at cell concentrations from 7.5 × 104 to 2.4 x 106 cells/well in the colorimetric microassay (11). A single concentration of 16 U/ml of natural murine CSF-1 was used in both assays to stimulate bone marrow cell proliferation. For the soft agarose assay, cells were grown in soft agarose on 35 mm petri plates for 7 days before colonies were counted using a Nikon microscope (40 X magnification). The experiment was perfon-ned in triplicate. For the coiorimetric microassay,cells were grown on rnicrotest plates for 3 days before the ability to metabolize 3-(4,5-dimetliylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (M'I'F) was determined. 20/xl of the MTT was added to each of the wells of the microtest plates. The cells were incubated for 4 h at 37 ° C. D.ufing this time, the MTT was reduced by mitoebondrial enzymesin the bone marrowcells to blue crystals of formazan. The formazan crystals were then dissolved by adding 100/~! of a 10% solution of sodium dodecyl sulfate in 0.01 N HCI and by further incubating the samples at 37o C for 16 h. The experiment was performed in quadruplicate. Points, means; bars, standard errors. examined for their ability to reduce M ' I T to formazan d y e and for the intensity of absorption which they produced. For purposes of comparison, the same range of bone marrow cell concentrations was examined in a soft agarose assay for their ability to form colonies. The data of a representative experiment are shown in Fig. 1. In agreement with previous studies (see Metcalf, 1984), the data show that the ability to form colonies in soft agarose was related in a dose-dependent fashion to the concentration of bone marrow ceils employed. The data also show that the reduction of M T T and thus the intensity of absorption was related in a similar dose-dependent fashion to the concentration of bone marrow cells employed. Thus, the soft agarose assay and the colorimetric microassay gave similar results.
26
Colorimetric assays are most accurate when the intensity of absorption is between 0.2 and 1 . 2 0 D . The intensities of absorption reported in Fig. 1 ranged from approximately 0.05 to 0.27 OD. However, it should be noted that these reported intensities are corrected values. The background intensities of absorption of wells in the microtest plates containing control cells plated in the absence of CSF-1 were always about 0 . 4 0 D and were automatically subtracted from the intensities of absorption in the test wells by our computer program. For example, for the range of cell concentrations expressed on Fig. 1 (7.5 × 104-2.4 × 106 cells/well), the intensi ties of absorption ranged from approximately 0.4 to 0.7. Thus, the values reported for the colorimetric microassay in Fig. 1 and in all subsequent figures lie within the appropriate absorption range (0.2-1.20D). Effect of CSF-I concentration on detection of bone marrow cell growth in the soft agarose assay and in the colorimetric microassay The growth of bone marrow cells in a soft agarose assay is proportional to the amount of growth factor to which the bone marrow cells are exposed. It was necessary to determine whether the colorimetric microassay would given comparable results. Bone marrow cells were grown either in the soft agarose assay or in the colorimetric microassay in the presence of concentrations of CSF-1 .ranging from 6.25 to 100 U/ml. The averaged data of four experiments are presented in Fig. 2. The data show that the growth of cells in the soft agarose assay is proportional to the concentration of CSF-1 employed. The growth of colonies in the colorimetric microassay was similarly proportional to the concentration of CSF-1 employed. Thus, the soft agarose assay and the colorimetric microassay gave comparable results. Calculation of CSF-! units in the colorimetric microassay A method of calculating CSF-1 activity has been established for the soft agarose assay (Koren et al., 1986). The method is based on the calculation of the linear regression line through the "linear' (actually log-linear) part of the titration curve. The titer of CSF-I is defined as the reciprocal of the dilution of CSF-1 required to give one bone
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Fig. 2. Comparisons of soft agarose assay and colorimetric microassay using different concentrations of CSF-1. Bone marrow cells were grown as described in Fig. I at a concentration of 7.5 x 104 cells/plate for the soft agarose assay (13) and at a concentration of 7.5 × 104 cells/well in the colorimetric microassay (n) in the presence of concentrations of CSF-I ranging from 6.25 to 100 U / m l . Data for the soft agarose assays are expressed as the aritl-,,netic m e a n s of four experiments performed in triplicate. Data for the colorimetric assays are expressed as the arithmetic m e a n s of four experiments performed in quadruplicate. Points, means; bars, standard errors.
marrow colony (extrapolated from the linear regression line). Such a definition of titer is easily applied to the colorimetric microassay. Fig. 3 presents a representative example of a titration of the laboratory standard CSF-1 preparation and of an unknown CSF-1 preparation Linear regression lines can be determined for the two CSF-1 preparations. By extrapolating each ol the linear regression lines to the abscissa, thc CSF-1 titer can be expressed as the reciprocal o! the dilution of CSF-1 which gives 0.00 absorbance. In this example, the laboratory standard has a titer of 752 U / m l in the colorimetric microassay. The unknown preparation has a titer of 34~ U / m l in the colorimetric microassay. Since th~ laboratory standard preparation has a known titeJ of 640 U / m l with the soft agarose assay, a simpk proportion can be set up and the titer of th~ unknown preparation can be predicted to be 29.U / m l . The actual titer on soft agarose of th( unknown preparation was determined to be 32(. U/trd, a difference of 11% and well within tht standard error of the assay. Alternatively, sinc~ the slopes for all samples for a given cytokine art essentially identical in a given assay, a GRIDL5
27
Effect of time of M T T reduction on the colorimetric microassay
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Fig. 3. Titration of a CSF-I preparation of unknown titer. Bone marrow cells at a cell concentration of 7.5 x 104 cells/well were grown as described in Fig. 1 for the colorimetric microassay in the presence of various dilutions of the laboratory standard CSF-1 preparation of known titer (121) or in the presence of a CSF-1 preparation of unknown titer (11). The data are expressed as the arithmetic means of a representative experiment performed in qaudruplicate. Points, means; bars, standard errors•
T h e c o l o r i m e t r i c a s s a y is d e p e n d e n t on the r e d u c t i o n o f M T T b y m i t o c h o n d r i a l enzymes to f o r m f o r m a z a n c r y s t a l s in the cells. In o r d e r to m e a s u r e the a m o u n t o f f o r m a z a n , the crystals m u s t b e d i s s o l v e d ( b y a d d i n g a s o l u t i o n of 10% s o d i u m d o d e c y l sulfate in 0.01 N HCI ( S D S / H C 1 solution)) b e f o r e the a m o u n t of f o r m a z a n can be e s t i m a t e d in an E I A reader. T w o c o m p e t i n g factors are involved in d e t e r m i n g the most app r o p r i a t e t i m e to r e a d the i n t e n s i t y of the formaTan: first, it takes s o m e time to dissolve the f o r m a z a n crystals a n d second, the intensity o f the f o r m a z a n d y e d e c r e a s e s with time after t r e a t m e n t with S D S / H C 1 . Fig. 4 p r e s e n t s the results of a n e x p e r i m e n t which c o m p a r e s the loss o f intensity of formazen with i n c r e a s i n g time after S D S / H C I treatment. T h e d a t a s h o w that the f o r m a z a n was most intense in its color 4 hours after t r e a t m e n t with SDS~-IC1. H o w e v e r , m i c r o s c o p i c e x a m i n a t i o n of the microtest p l a t e wells i n d i c a t e d that there were some
0.30
(grid search least squares fit for a n o n l i n e a r function) (Bevington, 1969) p r o g r a m can b e used to d e t e r m i n e the titer. U s i n g the f o r m u l a y = a (log x ) + b, the x i n t e r c e p t can b e d e t e r m i n e d as x i n t e r c e p t = 10 -(b/°). Since the titer is the reciprocal of the x intercept, titer = 10 (b/a). U s i n g the G R I D L S p r o g r a m , the titers of the l a b o r a t o r y s t a n d a r d p r e p a r a t i o n a n d of the u n k n o w n prep a r a t i o n c a n b e d e t e r m i n e d to b e 672 U / m l a n d 340 U / m l , respectively, in the c o l o r i m e t r i c assay. By simple p r o p o r t i o n a l i t y , the u n k n o w n p r e p a r a tion can b e p r e d i c t e d to have a titer o f 324 U / m l o n soft agarose. Since the a c t u a l titer o f the unk n o w n p r e p a r a t i o n o n soft a g a r o s e is 329 U / m l , the difference o f 1.5% is well w i t h i n the s t a n d a r d e r r o r of the assay. Thus, if a l a b o r a t o r y s t a n d a r d of k n o w n titer with the soft a g a r o s e assay is i n c l u d e d as a reference p r e p a r a t i o n , the relative titers can b e det e r m i n e d b y the c o l o r i m e t r i c m i c r o a s s a y a n d these titers can b e n o r m a l i z e d to the soft a g a r o s e titer of the l a b o r a t o r y s t a n d a r d p r e p a r a t i o n to give an e s t i m a t e d soft a g a r o s e titer for a n u n k n o w n C S F - 1 preparation.
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Fig. 4. Effect of time of MTT reduction on the colorimetric microassay. Bone marrow cells at a cell concentration of 7.5 x 10 4 cells/well were grown as described in Fig. 1 for the colorimetric microassay. After MTT was added to the cells, the cells were incubated for 4 h during which time the MTT was reduced by mitochondrial enzymes in the bone marrow cells to blue crystals of formazan. The formazan crystals were then dissolved by adding 100 pl of a 10% solution of sodium dodecyl sulfate in 0.01 N HC1. The samples were then incubated at 37°C for 4 h ( t ) , 16 h (11), or 40 h (A). The data are expressed as the arithmetic means of a representative experiments performed in quadruplicate. Points, means; bars. standard errors.
28 undissolved crystals present. The formazan was less intense in its color 16 h after treatment with SDS/HC1, a time when the formazan crystals were all dissolved. The curve generated 40 h after S D S / H C I addition showed that the intensity of the formazan continued to decrease with additional time after treatment with S D S / H C I . It should be noted that the titers of the CSF-1 (determined by extrapolation of the linear regression lines) were essentially identical for the three times of treatment with S D S / H C I (data not shown). Thus, although the intensity of the formazan dye decreased with increasing time of exposure of the formazan to SDS/HC1, the time of treatment with SDS/HC1 did not affect the determination of CSF-1 titer.
Measurement of r G M - C S F and 1L-3 actwities in the soft agarose assay and in the colorimetric microassay CSF-1 predominantly stimulates the gro ~¢th of macrophages. To test the general applicabiiity of the colorimetric microassay for detection of hematopoietic growth factors other than CSF-1, the soft agarose assay and the colorimetric microassay were evaluated for their abilities to detect
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Fig. 6. Comparisons of soft agarose assay and colorimetric microassay using different concentrations of IL-3. Bone marrow cells were grown as described in Fig. 1 at a cell concentration of 7.5 × 104 cells/plate for both the soft agarose assay (El) and at a concentration of 7.5 × 104 cells/well in the colorimetric microassay (11) in the presence of concentrations of IL-3 ranging from 3.1 to 100 U/ml. Data for the soft agarose assays are expressed as the arithmetic means of two experiments performed in quadruplicate. Points, means; bars. standard errors. G M - C S F and IL-3. GM-CSF stimulates the growth of granulocytes and macrophages while IL-3 stimulates the growth of erythroid progenitor cells, multipotential progenitor cells, eosinophils, granulocytes, macrophages, and lymphocytes (for review see Metcalf, 1984). Bone marrow cells were grown either in the soft agarose assay or in the colorimetric microassay in the presence of rGMCSF or IL-3. Fig. 5 presents the averaged data of five experiments using rGM-CSF. The data show that both the growth of colonies in the soft agarose assay and the growth of cells in the colorimetric microassay were similarly proportional to the concentration of r G M - C S F employed. Fig. 6 presents the averaged data of two experiments using IL-3. Again the data show that both the growth of colonies in the soft agaros6 assay and the growth of cells in the colorimetric microassay were similarly proportional to the concentration of IL-3 employed. Thus, the colorimetric microassay has a general a p p l i c a t i o n for the detection of hematopoietic growth factors.
Discussion
A colorimetric rnicroassay was developed by Mosmann (1983) which was based upon the
29 metabolism of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyhetrazolium bromide (MTT) to a blue formazan metabolite. The colorimetric microassay employed microtest plates and an ELISA Reader to measure IL-2- and mitogeninduced proliferation. Tada et al. (1986) further modified this microassay for measuring IL-2 activity. This report presents an adaptation of Tada et al.'s microassay method for the detection and quantitation of hematopoietic growth factors. The colorimetric microassay reported here employs 96-well microtest plates and permits the assay of many samples with minimal effort. The assays can be read directly in the wells of the microtest plates in a EIA Reader without transferring the samples. Using a personal computer and appropriate software (in this case a Macintosh computer and MacReader software), the data can be evaluated quickly. The assay has been shown to be useful for the detection and quantitation of a variety of hematopoietic growth factors including CSF-1, GM-CSF, and IL-3. The response of the bone marrow cells to these hematopoietic growth factors can be seen with the colorimetric microassay to be proportional to the amount of growth factor added over a wide range of growth factor concentrations. The data obtained with the colorimetric microassay are very similar to those generated with the soft agar assay of Bradley and Metcalf (1966). The colorimetric microassay permits the determination of the titers of hematopoietic growth factors and, by using a laboratory standard, one can normalize these titers to those generated by the soft agar assay. It should be noted while the formazan dye decays with time after treatment with 10% SDS in 0.01 N HC1, the titers determined by the colorimetric microassay are independent of the decay of the formazan dye. The colorimetric microassay has a number of advantages over the soft agar assay of Bradley and Metcalf (1966) for the detection of hematopoietic factor activity. First, the colorimetric microassay requires the expenditure of relatively smaller quantities of reagents which are often difficult to obtain. The soft agar assay is performed in 35 mm plastic petri dishes and, as such, it requires the expenditure of 2.5 ml of reagents. The colorimetric
microassay uses only 0.1 ml of reagents. Second, the colorimetric microassay is completed in a shorter period of time. The soft agar assay is dependent on the development of clones of cells into colonies and, as such, it requires an incubation period of 7-14 days. The colorimetric microassay can be read following a 3-day incubation period. Third, the colorimetric microassay is relatively less time consuming to evaluate. The soft agar assay is dependent on the microscopic examination of the plates to identify colonies as clones of 50 or more cells or as clones of cells with diameters of > 0.5 mm (Kriegler et al., 1987) and, as such, is time consuming, labor intensive, and somewhat subjective. The colorimetric microassay can be completed in minutes rather than hours. Finally, the colorimetric microassay can be used for the assay of large numbers of samples. One limitation of the colorimetric assay is that it cannot be used to distinguish between colonies and clusters. Another is that the colorimetric,-assay can not be used to type the colonies which have been stimulated to grow (i.e., macrophage colonies, granulocyte colonies, etc.). Recently, Kriegler et al. (1987) have developed a colorimetric tetrazolium assay to measure the activity of hematopoietic growth factors which stimulate the growth of macrophage progenitor cells. Their assay employed six-well cluster dishes (35 mm diameter) which required the investment of relatively large quantities of reagents. The method also required the manipulation of the samples before they could be read individually in a spectrophotometer. The colorimetric microassay reported here has several of the same advantages over the colorimetric assay of Kriegler et al. (1987) as it has over the soft agar assay of Bradley and Metcalf (1966): the colorimetric microassay uses smaller quantities of reagents; the colorimetric microassays can be read very quickly; and, the colorimetric microassay permits the assay of large number of samples in the same experiment. It should be noted that the soft agar or agarose assay has become the standard procedure for the determination of hematopoietic growth factor concentration and activity. Using the soft agar assay method, the activity of growth factors can be expressed in units. 1 U is defined as the amount of growth factor which must be present for the
30 development of one colony, as determined by extrapolating the linear regression line drawn through the linear portion of the growth factor titration curve. The colorimetric microassay gives a titration curve similar to that observed with the soft agar assay. Thus, the colorimetric microassay also permits an expression of growth factor titer in units by the extrapolation of its linear regression line. Using these definitions of units, the colorimetric microassay detects fewer units than the soft agar assay. For example, the laboratory standard has a titer of 188 U with the colorimetric microassay and 640 U with the soft agar assay. However, if a l a b o r a t o r y s t a n d a r d p r e p a r a t i o n of hematopoietic growth factor of known titer in the soft agar assay is included with each colorimetric microassay, the titers of each unknown can be normalized to the laboratory standard preparation and expressed according tn their relative expected titers in the soft agar assay. In summary, the colorimetric microassay can be used instead of the more cumbersome soft agar assay for a variety of experimental purposes. It is not anticipated that it could completely replace the soft agar assay. Because the colorimetric microassay does not examine clones microscopically, it does not permit the differentiation of different types of colonies (i.e., macrophage, granulocyte) which the soft agar assay permits.
Acknowledgements We thank Antonija Kotnik for her excellent technical assistance and William Law for his statistical assistance.
This study was supported by USPHS Grant CA26475 awarded by the National Cancer Institute, Department of Health and Human Services and by Grant IFN-3 awarded by the American Cancer Society.
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