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Table 2

Limiting Dilution Analysis of CD40-stimulated CD34 HPC

StimuliNbr CD34 HPC seeded/well*Percent wells with growthPercent wells containing DC
CD40L L-cells  100    1    0 
       
CD40L+ L-cells  100   84   36 
   10   27   10 
    1    6    3 
       
GM–CSF/TNF  100  100  100 
   10   38   27 
    1   16   12 
Limiting dilution assay. CD34HPC were seeded at a density of 1, 10, or 100 cells/well in round-bottomed microtiter plates at a final volume of 100 μ1 on irradiated CD40L+ cells or with GM–CSF (100 ng/ml)/ TNF (2.5 ng/ml). After 12 d of culture, the percentage of total wells with cell growth and the percentage of total wells containing DC (as determined from morphology in culture plates examined with an inverted microscope) were scored. For each condition, a total number of 60 wells were seeded with 100 CD34HPC/well and 600 wells were seeded with 10 or 1 CD34HPC/well. One plate containing CD40L negative fibroblastic L cells was seeded with 100 CD34HPC/well and used as a negative control. *For 1 and 10 cells/well, 600 wells were seeded. For 100 cells/wells, 60 wells were seeded. 
StimuliNbr CD34 HPC seeded/well*Percent wells with growthPercent wells containing DC
CD40L L-cells  100    1    0 
       
CD40L+ L-cells  100   84   36 
   10   27   10 
    1    6    3 
       
GM–CSF/TNF  100  100  100 
   10   38   27 
    1   16   12 
Limiting dilution assay. CD34HPC were seeded at a density of 1, 10, or 100 cells/well in round-bottomed microtiter plates at a final volume of 100 μ1 on irradiated CD40L+ cells or with GM–CSF (100 ng/ml)/ TNF (2.5 ng/ml). After 12 d of culture, the percentage of total wells with cell growth and the percentage of total wells containing DC (as determined from morphology in culture plates examined with an inverted microscope) were scored. For each condition, a total number of 60 wells were seeded with 100 CD34HPC/well and 600 wells were seeded with 10 or 1 CD34HPC/well. One plate containing CD40L negative fibroblastic L cells was seeded with 100 CD34HPC/well and used as a negative control. *For 1 and 10 cells/well, 600 wells were seeded. For 100 cells/wells, 60 wells were seeded. 
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