Growth factor requirements for short-term growth of ELM cells in the presence or absence of stroma. (A) Effect of growth factors on the growth of ELM cells in serum-containing medium after removal from MS5 stroma cells. A total of 104 ELM cells were plated in 96-well plates in medium containing 8% horse serum in the presence of increasing amounts of IGF-1, insulin, or SCF. Proliferation under these conditions was assayed by the MTT method and data shown are mean values from three independent experiments (12 cultures) with standard errors. The data shown are for the minimum amounts of growth factors found to give the maximal stimulation (SCF, 10 ng/mL; IGF-1, 100 ng/mL; insulin, 10 μg/mL). (B) Effect of growth factors and neutralizing antibodies on the growth of ELM cells in serum-free medium or 4% serum. A total of 104 ELM cells were plated in 96-well plates in serum-free medium in the presence of 100 ng/mL IGF-1 or 10 ng/mL SCF and 30 μg of neutralizing antibodies to IGF-1 (I1Ab) or the SCF-R (ACK2). Proliferation under these conditions was assayed by the MTT method and data shown are mean values from three independent experiments (12 cultures) with standard errors. (C) Effect of antibodies against IGF-1, SCF, or integrins on the growth of ELM cells on MS5 stroma cells. A total of 100 ELM cells were plated on 200 MS5 cells in wells in Terasaki plates and incubated for 5 days in the presence of 30 μg/mL neutralizing antibodies to SCF-R (ACK-2), IGF-1 (I1Ab), or α4 integrin (α4Ab). The right-hand panel shows the growth of ELM cells without stroma. The results are the number of cells in the wells relative to the number on stroma alone and represent the mean of three independent experiments (24 cultures) with standard errors.
