colorimetric determination of inhibition of hematopoietic progenitor cells in soft agar

Journal of Immunological Methods 244 (2000) 49–58www.elsevier.nl / locate / jim

Colorimetric determination of inhibition of hematopoieticprogenitor cells in soft agar

*Daniel Horowitz , Andrew G. KingDepartment of Molecular Virology and Host Defense, SmithKline Beecham Pharmaceuticals, Collegeville, PA 19426-0989, USA

Received 16 November 1999; received in revised form 5 June 2000; accepted 6 June 2000

Abstract

In vitro colony forming unit (CFU) assays have been used to measure the effects of compounds that regulate the growth ofhematopoietic progenitor cells. These assays are time consuming and subjective and are therefore not amenable to highthroughput of large numbers of compounds. Here we have shown that the traditional murine bone marrow CFU assay can bemodified into a robust non-subjective colorimetric assay format. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) was added after colony formation in an agar based 96-well plate culture system. Optical density correlatedwith increasing cell input concentrations in the presence of growth factor. The linearity of this response was equivalent to thestandard CFU assay. Several hematopoietic inhibitors were tested in both assays. Effects on colony number and size werecompared to optical density. Compounds that reduced colony numbers with little effect on colony size had identical IC50

values in both the colorimetric assay and CFU assay. The IC values of compounds that also decreased colony size did not50

correlate in the two assays. These results demonstrate the utility of the colorimetric assay to rapidly screen for compoundsthat specifically inhibit hematopoietic progenitor cell colony formation in vitro. 2000 Elsevier Science B.V. All rightsreserved.

Keywords: Hematopoiesis; Growth factor; Colony formation; MTT

Abbreviations: CFU, colony-forming unit; MTT, 3-[4,5-Di- 1. Introductionmethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; IC , 50%50

inhibitory concentration; M-CSF, macrophage colony stimulatingMature blood cells are produced from the differen-factor; G-CSF, granulocyte colony stimulating factor; GM-CSF,

tiation of hematopoietic progenitor cells in the bonegranulocyte-macrophage colony stimulating factor; TGFb, trans-forming growth factor beta; IFNg, gamma interferon; AZT, 39- marrow (Morstyn & Burgess, 1988; Quesenberry,azido-39-deoxythymide; Ara-C, cytosar; 5-FU, 5-fluorouracil; 1992; Gordon, 1994). Progenitors can be assayed inSDS, sodium dodecyl sulfate vitro by incubation of cells in a semi-solid medium

*Corresponding author. Tel.: 11-610-917-6784; fax: 11-610-with factors capable of supporting growth and dif-917-4178.ferentiation (Bradley & Metcalf, 1966). Cells ofE-mail address: daniel [email protected] (D.

]Horowitz). different lineages can be detected with these assays

0022-1759/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0022-1759( 00 )00253-2

50 D. Horowitz, A.G. King / Journal of Immunological Methods 244 (2000) 49 –58

depending on the growth factors used. Progenitor lated human CD341 cells cultured in liquid mediumcells of the myeloid lineage produce granulocytes measured cytotoxic drug growth inhibition, however,and macrophages and can form colonies in soft agar the reported IC values for hematopoietic growth50

in response to growth factors such as macrophage factor-induced clonogenic (granulocyte–macrophagecolony stimulating factor (M-CSF), granulocyte colonies) and liquid expansion cultures did not matchcolony stimulating factor (G-CSF) and granulocyte– (Leglise et al., 1996).macrophage colony stimulating factor (GM-CSF) In the present study, modification of the MTT(Heyworth & Spooncer, 1993). colorimetric assay (Mosmann, 1983) was developed

The CFU assay has been used to screen com- in which 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-pounds that can affect the formation and growth of tetrazolium bromide (MTT) was added directly tohematopoietic cells (Deldar & Stevens, 1993; Schoe- bone marrow cells with hematopoietic growth factorsters et al., 1995). Drugs used clinically for various in soft agar after colony formation. To determine ifdiseases may have adverse affects on the hemato- this colorimetric assay could be used as a correlatepoietic system. For example, during a course of assay, colony counts were compared to thecytotoxic therapy for treatment of cancer, hemato- colorimetric readout using different growth factors.poietic cells as well as malignant cells can be killed Conditions were optimized using M-CSF as a repre-by the treatment. This can result in loss and delayed sentative hematopoietic growth factor. Transformingrecovery of mature blood cells leading to possible growth factor beta 1 (TGFb1), Interferon gammainfections or hemorrhage (Evans, 1988; Gale, 1988). (IFNg), 39-azido-39-deoxythymide (AZT), ganci-In addition to cytotoxic compounds, several proteins clovir, cytosar (ara-C) and 5-fluorouracil (5-FU)including TGFb and IFNg have been shown to block were used to inhibit colony formation. TGFb1 andhematopoietic cell proliferation (Graham, 1997) and IFNg are cytokines that inhibit colony formation inare important for the regulation of hematopoiesis. response to certain growth factors (Ohta et al., 1987;

Due to the labor intensiveness of the CFU assay, a Battistini et al., 1990; Graham, 1997). The chemo-more rapid assay would allow screening of a larger therapeutic antimetabolite agents 5-FU and ARA-Cnumber of compounds. Colony assays have been kill tumor and hematopoietic cells by inhibitingdeveloped with tumor cell lines using colorimetric DNA synthesis (Marsh, 1976; Yeager et al., 1983;readout of viable cells. A colorimetric determination Gale, 1988). Myelosuppression is a major side effectto assess the clonogenic capacity and chemosensitivi- of antiviral drug therapy with AZT and ganciclovir.ty of human leukemic cell lines in response to In vitro assays have demonstrated direct inhibition ofgrowth modulators correlated very well with colony hematopoietic colony formation by these compoundscounting (Schweitzer et al., 1993). In fact, the (Scheding et al., 1994; Dornsife & Averett, 1996). Incolorimetric determination was shown to be more this study we determined the IC values for these50

sensitive and a greater predictor of in vivo sensitivity compounds in the colorimetric assay using M-CSF oror resistance (Abe et al., 1994). In the bone marrow, GM-CSF to determine the utility of this assay formatthere is a heterogeneous population of hematopoietic as a more rapid screen of potential hematopoieticcells at different stages of differentiation (Graham & inhibitors.Pragnell, 1992). In a CFU assay colonies of varioussizes and cell types may be formed depending ongrowth factor combinations used. Even with a single 2. Materials and methodsgrowth factor there can be heterogeneity in colonyformation. A colorimetric assay, which detects total 2.1. Micecell viability, may give different results with aheterogeneous population such as colonies formed Female mice (C57BL/63DBA/2)F1(B6D2F1)from bone marrow compared to colonies formed from Harlan Sprague Dawley were used between 7from clonal populations. and 16 weeks old. Mice were provided with acidified

An MTT-based colorimetric assay utilizing iso- water and housed along with sentinel mice that were

D. Horowitz, A.G. King / Journal of Immunological Methods 244 (2000) 49 –58 51

routinely screened and shown to be pathogen free. incubated at 378C, 6–8% CO . For the CFU assay,2

The Institutional Animal Care and Use Committee of colony formation was analyzed after 7 days using aSmithKline Beecham Pharmaceuticals approved pro- Zeiss inverted stereo microscope. Colonies werecedures involving the use of laboratory animals. defined as groups of 50 or more cells. Large colonies

were defined as greater than 200–300 cells. For the2.2. Media colorimetric assay, 50 ug of MTT (3-[4,5-Di-

methlythiazol-2-yl]-2,5-diphenyltetrazolium bro-McCOY’s 5a media, modified with L-glutamine mide; Thiazoyl blue) (Sigma) in phosphate buffered

and supplemented with 100 U penicillin, 100 ug/ml saline was added to each well. After 4–6 h, incuba-streptomycin, 0.6X MEM vitamin solution, 1X tion at 378C, 6–10% CO , 75 uL of 10% SDS2

MEM sodium pyruvate, 0.8X MEM essential amino (sodium dodecyl sulfate) in 0.01 N HCl was added toacids, 0.6X MEM non-essential amino acids, 0.05% each well. Plates were incubated 18–48 h at 378C,sodium bicarbonate and 15% heat inactivated fetal 6–8% CO to solubilize the formazan dye. Optical2

bovine serum were used. All media reagents were density was measured using a Biotek Instrumentspurchased from GIBCO BRL except fetal bovine EL-312 Biokinetic plate reader with a 570 nm filterserum (Hyclone, Logan, UT). and a 750 nm reference filter. The IC was defined50

as the concentration at half of the response at the2.3. Bone marrow preparation maximum concentration tested and the response with

no compound. Analysis of IC values for both50

Animals were killed by CO asphyxiation. Femurs assays were calculated using a four parameter non-2were flushed with 2.5 ml of culture medium and linear regression with Statistica software.

diluted 1:10 in culture medium. Cell suspensionswere incubated in tissue culture flasks for 90 min at378C. Non-adherent cells were removed and spun for10 min at 1800–1900 RPM in a Beckman GS-6Rcentrifuge. Pellets were resuspended in culturemedium with a syringe and a 25 gauge needle tocreate a single cell suspension. Cells were countedusing a Technicon H1 blood analyzer.

2.4. CFU and colorimetric assays

Human TGFb1 (R1D systems), murine IFNg

(R1D systems), 5-FU (Hoffman-LaRoche Inc.),ARA-C (Upjohn), AZT (Sigma) or ganciclovir (Hof-

fman-LaRoche) was added to Falcon 12-well platesfor the CFU assay or Nunc 96-well plates for thecolorimetric assay. Cells were mixed with Agar(Difco Laboratories) in culture medium (0.3% final

Fig. 1. Colorimetric response with growth factors, 50,000 (M-concentration) with or without growth factors and CSF and GM-CSF) or 100,000 (G-CSF) non-adherent boneadded to plates. Each micro-titer plate included wells marrow cells /ml were mixed with 20 ng/ml growth factor andwithout growth factor as a negative control for that set-up in a CFU or colorimetric assay as described in Section 2.

After 5 days (M-CSF and GM-CSF) or 7 days (G-CSF) incuba-plate. Murine M-CSF and GM-CSF were purchased tion at 378C, 6% CO , optical density was determined for the2from R1D systems. Human G-CSF (Neupogen )

colorimetric assay. Colonies were counted after 7 days. Threewas purchased from Hanna Pharmaceuticals. The groups of experiments are shown. For each growth factor bothfinal volumes for the CFU assay and the colorimetric assays were done from the same pool of marrow. Each barassay were 500 and 200 uL respectively. Plates were represents the mean of three wells6S.E.M.


3. Results

3.1. Colorimetric response with M-CSF, G-CSF orGM-CSF

Three separate CFU and colorimetric assays weresetup with M-CSF, G-CSF or GM-CSF (Fig. 1).Total colonies were compared to optical densitydetermined after MTT conversion. In the experimentperformed with M-CSF, the mean optical densitieswere 0.580 in wells with M-CSF and 0.168 in wellswithout M-CSF. The mean CFU/well were 39 withM-CSF and 0 without M-CSF. The mean opticaldensities were 0.324 with G-CSF and 0.271 without Fig. 2. Optimum cell incubation time for colorimetric assay.

Non-adherent bone marrow cells were incubated with 10 ng/mlG-CSF. The mean CFU/well were 7 with G-CSF.M-CSF for 4 (d), 5 (♦), 6 (m) or 7 (j) days prior to MTTThere were no wells without growth factor. Theaddition. The reaction was stopped after 4–6 h. and plates were

mean optical densities were 0.745 with GM-CSF and read as described in Section 2. Each point represents the mean0.116 without GM-CSF. The mean CFU/well were optical density of 3-wells with M-CSF minus the optical density39 with GM-CSF and 0 without GM-CSF. For both from the corresponding cell concentration without M-

CSF6S.E.M.M-CSF and GM-CSF the optical density and CFUvalues were significantly higher in wells with growthfactor compared to wells without growth factor (Pvalue ,0.01). The optical density in the colorimetric response was linear in both assays from 12,500assay using G-CSF was not significantly higher than cells /ml to 100,000 cells /ml. The slope of both dosebackground. responses were also equivalent indicating that colony

number increased at the same rate as optical density3.2. Determination of optimal cell concentration (Fig. 3).and incubation time for the colorimetric assayusing M-CSF

Because of the lack of response in the colorimetricassay with G-CSF, only M-CSF and GM-CSF wereused in subsequent experiments. M-CSF was used tooptimize assay conditions. Non-adherent bone mar-row cells were incubated with 10 ng/ml of M-CSFin 96-well plates at 12,500–100,000 cells /ml. Plateswere incubated from 4 to 7 days before MTTaddition. The peak colony response occurred at day 7in cultures with 50,000 cells plated. The opticaldensity was slightly lower with 100,000 cells on day7 but the response was linear from 12,500 cells /mlto 50,000 cells /ml (Fig. 2).

The standard hematopoietic progenitor CFU assay Fig. 3. Cell dose response in the colorimetric (j) and CFU (h)assays. Non-adherent bone marrow cells were incubated with 20is incubated from 7 to 12 days before colonies areng/ml M-CSF for 7 days. Colonies were counted in the CFUcounted (Heyworth & Spooncer, 1993). In anotherassay and MTT was added to the colorimetric assay as described

experiment a cell dose response in the standard CFU in Section 2. Each point represents the mean of threeassay was compared to the colorimetric assay at 7 values6S.E.M. Optical density is the value of M-CSF minus thedays using 20 ng/ml M-CSF. In this experiment the value from the corresponding cell concentration without M-CSF.


nM616, CFU5140 nM68). There was completeinhibition in both assays at 329 nM (Fig. 5B). Bothof these compounds appeared to affect mainly colonynumber since both small and large colonies declinedas total CFU decreased.

3.5. Inhibition with anti-viral agents in thecolorimetric and CFU assays using M-CSF

The IC value for AZT was 26 uM62 in the50

CFU assay and 14 uM62 in the colorimetric assay(Fig. 5C). AZT appeared to affect colony size morethan colony number. Large colonies were completelyFig. 4. M-CSF dose response. 50,000 non-adherent bone marrowinhibited. However the inhibition of total CFU at thecells /ml were incubated with different concentrations of M-CSF

for 7 days in the colorimetric assay and CFU assay as described in highest concentration tested was about 60%. In theSection 2. Each point represents the mean percent from two colorimetric assay the maximum inhibition wasexperiments of values for total CFU (small CFU (solid bar), large

about 85%. Distinctions of only large and smallCFU (white bar)) or optical density (h) at 40 ng/ml M-CSF. Thecolonies were included, however it is more likelymean number of colonies at 40 ng/ml M-CSF was 21 smallthat there is a continuum of colony size. Althoughcolonies, 16 large colonies and 36 total colonies. The mean net

optical density at 40 ng/ml was 0.712. the number of small colonies decreased slightly asAZT concentrations increased, the number of cellsper colony may be decreased resulting in a lower

3.3. M-CSF dose response in colorimetric andoptical density. Ganciclovir had equivalent IC50CFU assaysvalues in both assays (colorimetric551 uM64,CFU554 uM64) and completely inhibited colony

A comparison of the M-CSF dose response be-formation at concentrations greater than 90 uM (Fig.

tween the CFU assay and the colorimetric assay was5D).

done using 50,000 cells /ml and an incubation timefor both assays of 7 days. Doses of M-CSF rangedfrom 0.16 ng/ml to 40 ng/ml. (Fig. 4). As the 3.6. Inhibition with cytokines in colorimetric andM-CSF concentrations decreased, the optical density CFU assays using M-CSFdecreased faster than total CFU numbers. The ratioof large to small colonies decreased as growth factor TGFb1 and IFNg were tested at concentrationsconcentration decreased. Since the colorimetric assay ranging from 0.02 to 800 ng/ml. For TGFb1 themeasures total viable cells, decreases in colony size IC value in the CFU assay was 2.2 ng/ml60.5. In50

as well as total colony number will result in lower contrast, the IC value could not be calculated50

optical densities. accurately for the colorimetric assay due to variationin the dose response. In the CFU assay the maximum

3.4. Inhibition with anti-cancer agents in the inhibition by TGFb was about 40%. In thecolorimetric and CFU assays using M-CSF. colorimetric assay the optical density dropped and

then started to rise after 100 ng/ml (Fig. 5E). The5-FU was tested at concentrations ranging from IC value for IFNg was about 10-fold higher in the50

240 to 3844 nM. The IC value for 5-FU was CFU assay (19.4 ng/ml611.6) than in the colorimet-50

almost equivalent in both assays (colorimetric5730 ric assay (2.1 ng/ml60.7). The maximum concen-nM661, CFU5853 nM615). There was complete tration of INFg tested gave 50% inhibition in theinhibition in both assays at concentrations of 5-FU CFU assay and about 20% inhibition in thegreater than 1000 nM (Fig. 5A). Ara-C had equiva- colorimetric assay. As in the AZT assay there waslent IC values in both assays (colorimetric5132 more of an effect on colony size than colony number50


Fig. 5. Dose response in M-CSF CFU and colorimetric assays. 50,000 non-adherent bone marrow cells /ml were incubated with 20 ng/ml ofM-CSF for 7 days in the colorimetric assay and CFU assay as described in Section 2. Each point represents the percent of values for smallCFU (black bar), large CFU (white bar) or net optical density (h) without compound. The mean number of colonies per well withoutcompound were 33 small colonies, 8 large colonies and 41 total colonies. The mean net optical density was 0.748 nm. IC values were50

based on total colonies and optical density. Each line represents the mean or composite of two to six experiments.


agent ganciclovir were tested again in both assaysusing GM-CSF, an earlier acting growth factor thanM-CSF. ARA-C had equivalent IC values in the50

GM-CSF assay (colorimetric580 nM66; CFU574nM64). Ganciclovir also had equivalent IC values50

(colorimetric534 uM65; CFU534 uM62). TheIC values of both compounds were about half of50

the IC value using M-CSF, however the pattern of50

inhibition was similar.

4. Discussion

A microtiter colorimetric assay was developed asan alternative to the standard CFU assay to addressissues of subjectivity and throughput inherent to themanual enumeration of CFU by microscopy. TheCFU assay is a useful way to identify potentialhematopoietic inhibitors, however it is not easilyadaptable to a rapid screening format. The CFUassay requires large area well culture plates, whichmakes it difficult to automate. In addition, micro-scopic quantification of hematopoietic colonies istime consuming and requires specialized training.Colony counting has been shown to vary betweenlabs even with good quality control of reagents. Thisappears to be due to differences in the scoring ofcolonies (Lumley et al., 1999; Lamana et al., 1999).A more rapid assay with a non-subjective readoutwould address these issues.Fig. 6. Dose response in GM-CSF CFU and colorimetric assays.

The microtiter colorimetric assay described herein50,000 non-adherent bone marrow cells /ml were incubated with20 ng/ml of GM-CSF for 7 days in the colorimetric assay and measures MTT conversion after addition to growthCFU assay as described in Section 2. Each point represents the factor-stimulated hematopoietic colonies in soft agar.percent of values for small CFU (black bar), large CFU (white

The assay correlated with colony inhibition in 12-bar) or net optical density (h) without compound. The meanwell plates scored microscopically by colony count-number of colonies per well without compound were 24 smalling. Although colorimetric assays correlating colonycolonies, six large colonies and 30 total colonies. The mean net

optical density was 0.510 nm. IC values were based on total growth with a colorimetric readout have been de-50

colonies and optical density. Each line represents the mean of two veloped with tumor cells, this approach has not beenexperiments.

described previously with bone marrow cells. Analy-sis of murine bone marrow cultures stimulated with

resulting in greater inhibition detected in the M-CSF, G-CSF or GM-CSF demonstrates that thecolorimetric assay (Fig. 5F). colorimetric readout was dependent on colony for-

mation. There were significant increases in optical3.7. Inhibition in colorimetric and CFU assays density with M-CSF and GM-CSF compared to wellsusing GM-CSF without growth factor. There was no change in color

with G-CSF. However, there were very few coloniesThe anti-cancer agent ARA-C and the anti-viral in the G-CSF CFU assay. It has been reported that


G-CSF tends to produce few colonies in an agar for both compounds were about half of the IC50

based murine system (Metcalf & Nicola, 1983). value with M-CSF.Therefore G-CSF was not used to compare inhibition The inhibitory cytokine IFNg had an IC value50

between the colony and the colorimetric assay. about 10-fold lower in the MTT assay than in theThe M-CSF dose response was compared in both CFU assay. The IC value for TGFb in the50

assays. In the colorimetric assay the peak response colorimetric assay could not be accurately deter-was 10 ng/ml M-CSF whereas in the conventional mined due a variable dose response. Large coloniesCFU format, 5 ng/ml M-CSF was sufficient to were completely inhibited with IFNg, while smallstimulate peak numbers of CFU. Colony size analy- colonies where only slightly inhibited. Both TGFb

sis showed that the number of large colonies de- and IFNg have been shown to affect the rate of cellcreased and the number of small colonies increased, growth as well as colony formation (Kramer et al.,however, the total number of colonies remained 1994; Shiohara et al., 1994). The reduction in theconstant in both assay formats at M-CSF doses of rate of growth of colonies would result in fewer large5–40 ng/ml. The colorimetric response dropped colonies relative to total after 7 days. TGFb has beenfaster than total CFU as M-CSF concentrations reported to have differential effects on hematopoieticdropped. Since the colorimetric assay measures cells. It can inhibit progenitors but can stimulateresponses from the total number of cells, a decrease proliferation of mature cells (Ruscetti et al., 1993).in colony size as well as number would account for a At 7 days, colonies may contain enough mature cellsdecrease in the colorimetric response. that could be stimulated by TGFb. The combination

The degree of inhibition, as determined by IC of colony inhibition and stimulation of mature cells50

values, was measured for several known hemato- may explain the experimental variation observed inpoietic inhibitors in both the CFU and colorimetric the colorimetric assay.assays. The anti-cancer agents 5-FU and ARA-C and The apparent lack of correlation between thethe anti-viral ganciclovir each had equivalent IC colony and colorimetric assay with cytokine in-50

values in both assays. The IC value for the anti- hibitors such as IFNg and TGFb may involve several50

viral AZT was lower in the colorimetric assay (14 factors. TGFb may have multiple effects on differentuM) than in the CFU assay (26 uM); however, these cell types contained within the bone marrow popula-values were within 2-fold of each other. With 5-FU, tion used for these assay systems. A combination ofARA-C and ganciclovir the large colonies appeared suppressive activity and stimulatory activity onto be inhibited proportionate to the small colonies. different cell types would result in unusual doseHowever, AZT completely inhibited large colonies, response curves as defined in the colorimetric assaywhile small colonies were reduced by around 60%. since this assay measures total cellular mitochondrialFrom these experiments it cannot be determined activity. IFNg decreased colony size with only awhether AZT had more of an effect on the formation 40% decrease in total colony number. This wouldof large colonies over small colonies or whether result in a decrease in total viable cells and athere was an overall reduction in colony size. decrease in MTT conversion. In contrast, the activityHowever, a differential reduction in colony size and in the colony assay is specific for CSF-responsivenumber would explain differences in inhibition as progenitors.measured by total cell viability (colorimetric assay) Progenitor cell inhibitors or toxic agents with noand colony number (CFU assay). inherent hematopoietic properties such as the anti-

We have shown that the formation of GM-CSF cancer and anti-viral agents tested herein, resulted inresponsive colonies can also be measured with the equivalent inhibition in the CFU and the colorimetriccolorimetric assay. Ara-c and ganciclovir were used assays with M-CSF and GM-CSF. Because the typesto determine if the colorimetric assay could be of cells in the colonies produced in these assays werecorrelated with GM-CSF responsive colony inhibi- not examined, it could not be determined whattion. Each of these compounds had equivalent IC effects these compounds had on specific lineages.50

values in both assays. The IC value with GM-CSF Although murine cells were used in these experi-50


Gordon, M.Y., 1994. Physiology and function of the haemopoieticments, it has been reported that the anti-viral andmicroenvironment. Br. J. Haematol. 86, 241.anti-cancer compounds used also inhibited colony

Graham, G.J., Pragnell, I.B., 1992. The haemopoietic stem cell:formation from human marrow (Leglise et al., 1996). properties and control mechanisms. Semin. Cell Biol. 3, 423.Cytotoxic compounds had different IC values on Graham, G.J., 1997. Growth inhibitors in haemopoiesis and50

different hematopoietic subsets. The IC values in leukaemogenesis. Baillieres. Clin. Haematol. 10, 539.50Heyworth, C.M., Spooncer, E., 1993. In vitro clonal assays forthe colony assays were shown to correlate with in

murine multipotential and lineage restricted myeloidvivo hematological toxicity (Dornsife & Averett,progenitors cells. In: Testa, N.G., Molineux, G. (Eds.),

1996). These results indicate that the colorimetric Haemopoiesis. Oxford University Press, p. 37.assay could be utilized as a rapid screening assay for Kramer, I.M., Patel, R., Spargo, D., Riley, P., 1994. Initiation ofthe identification of small molecule hematopoietic growth inhibition by TGF beta 1 is unlikely to occur in G1. J.

Cell Sci. 107, 3469.inhibitors in place of the traditional CFU assay. TheLamana, M., Albella, B., Rodriguez, F., Regidor, C., Bueren, J.A.,colorimetric readout should also reduce lab to lab

1999. Conclusions of a national multicenter intercomparativevariation seen in the CFU assay with the subjective study of in vitro cultures of human hematopoietic progenitors.read out of colony identification. Bone Marrow Transplantation 23, 373.

Leglise, M.C., Detailly, P.D., Vignot, J.L., Lebot, M.A., Leroux,A.M., Riche, C., 1996. A cellular model for drug interactionson hematopoiesis-the use of human umbilical cord bloodprogenitors as a model for the study of drug-related myelosup-Acknowledgementspression of normal hematopoiesis. Cell Biol. Toxicol. 12, 39.

Lumley, M.A., Burton, A., Billingham, L.J., McDonald, D.F.,The authors would like to thank Robert Gagnon Czarnecka, H.M., Milligan, D.W., 1999. Quality assurance of

CFU-GM assays: inter-laboratory variation despite standardfor help with statistical analysis and Javier Garcia forreagents. Eur. J. Haematol. 62, 32.excellent technical assistance.

Marsh, J.C., 1976. The effects of cancer chemotherapeutic agentson normal hematopoietic precursor cells: a review. [Review][206 refs]. Cancer Res. 36, 1853.

Metcalf, D., Nicola, N.A., 1983. Proliferative effects of purifiedReferences granulocyte colony-stimulating factor (G-CSF) on normal

mouse hemopoietic cells. J. Cellular Physiol. 198–206.Morstyn, G., Burgess, A.W., 1988. Hemopoietic growth factors: aAbe, R., Ueo, H., Akiyoshi, T., 1994. Evaluation of MTT assay in

review. Cancer Res. 48, 5624.agarose for chemosensitivity testing of human cancers: com-Mosmann, T., 1983. Rapid colorimetric assay for cellular growthparison with MTT assay. Oncology 51, 416.

and survival: application to proliferation and cytotoxicityBattistini, A., Affabris, E., Fiorucci, G., Coccia, E.M., Romeo, G.,assays. J. Immunological Methods 65, 55.Marziali, G., Rossi, G.B., 1990. Spectrum of biological

Ohta, M., Greenberger, J.S., Anklesaria, P., Bassols, A., Mas-activity of interferons. [Review] [324 refs]. Annali dell Istitutosague, J., 1987. Two forms of transforming growth factor-betaSuperiore di Sanita 26, 227.distinguished by multipotential haematopoietic progenitorBradley, T.R., Metcalf, D., 1966. The growth of mouse bonecells. Nature 329, 539.marrow cells in vitro. Aust. J. Exp. Biol. Med. Sci. 44, 287.

Quesenberry, P.J., 1992. Stroma-dependent hematolymphopoieticDeldar, A., Stevens, C.E., 1993. Development and application ofstem cells. [Review] [130 refs]. Curr. Top. Microbiol. Im-in vitro models of hematopoiesis to drug development. Tox-munol. 177, 151.icol. Pathol. 21, 231.

Ruscetti, F., Varesio, L., Ochoa, A., Ortaldo, J., 1993. PleiotropicDornsife, R.E., Averett, D.R., 1996. In vitro potency of inhibitioneffects of transforming growth factor-beta on cells of theby antiviral drugs of hematopoietic progenitor colony forma-immune system. [Review] [56 refs]. Ann. NY Acad. Sci. 685,tion correlates with exposure at hemotoxic levels in human488.immunodeficiency virus-positive humans. Antimicrob.Agents

Scheding, S., Media, J.E., Nakeff, A., 1994. Acute toxic effects ofChemother. 40, 514.39-azido-39-deoxythymidine (AZT) on normal and regenerat-Evans, W.E., 1988. Clinical pharmacodynamics of anticancering murine hematopoiesis. Exper. Hematol. 22, 60.drugs: a basis for extending the concept of dose-intensity. Blut

Schoeters, G.R., Vanderplaetse, F., Leppens, H., Vanvlasselaer, P.,56, 241.Vandenheuvel, R., 1995. Haemopoietic and osteogenic toxicityGale, R.P., 1988. Myelosuppressive effects of antineoplastictesting in vitro using murine bone marrow cultures. Toxicolo-chemotherapy. In: Testa, N.G., Gale, R.P. (Eds.), Hemato-gy in vitro 9, 421.poiesis. Long-Term Effects of Chemotherapy and Radiation.

Schweitzer, C.M., van-de, L.A., Jonkhoff, A.R., Ossenkoppele,Marcel Dekker, New York, p. 63.


G.J., Huijgens, P.C., Drager, A.M., Broekhoven, M.G., and stem cell factor. [Review] [71 refs]. Leukemia andLangenhuijsen, M.M., 1993. Spectrophotometric determination Lymphoma 14, 203.of clonogenic capacity of leukemic cells in a semisolid Yeager, A.M., Levin, J., Levin, F.C., 1983. The effects of 5-microtiter culture system. Exp. Hematol. 21, 573. fluorouracil on hematopoiesis: studies of murine

Shiohara, M., Koike, K., Nakahata, T., Komiyama, A., 1994. megakaryocyte-CFC, granulocyte-macrophage-CFC, andHematopoietic progenitors and synergism of interferon-gamma peripheral blood cell levels. Exp. Hematol. 11, 944.

colorimetric determination of inhibition of hematopoietic progenitor cells in soft agar

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