G-CSF Bioassay through HL-60 Cell Line Treated with Retinoic Acid and DMSO

The use of accurate and reliable bioassay methods for recombinant proteins is a key element in the production and utilization of such proteins. In this research we have tried to present a reliable method for the G-CSF bioassay. HL-60 cells belonged to human leukemia cells are capable to be differentiated into macrophages or neutrophils. When stimulated by different agents like DMSO, RA and cAMP, HL-60 cells are differentiated into neutrophils, while interferon and phorbol ester differentiate them into macrophages. In this study HL-60 cells were first cultured in flasks. Then 2 x 105 cell/ml of them were selected and 100 ul of this cell suspension was transferred to a 96-well microplate. Then proper dilution of DMSO and RA was prepared and added to the cell suspension (1.3% and 0.1 flM, respectively) and incubated at 37°C and 5% C02 atmosphere. Using RA and DMSO, the cell were transferred from the proliferative phase to the differentiative phase. Then various concentrations of GCSF were added to these treated cells and incubatedat 37°C and 5% C02 atmosphere for two days. The MTT assay was used to measure the reversal rate of the cells from the differentiative to the proliferative phase. The cell proliferation was evaluated by mitochondrial S.T.R. enzme system through the reduction of MTT to Furmazan. The results showed that this method is reliable for the G-CSF bioassay.