Assay for functional activity of GM-CSF produced by A20-transduced cells
9. Pipet 50 pl conditioned medium from GM-CSF-transduced cells growing in step 8 into triplicate wells of a 96-well tissue culture plate and dilute to 1:2 using complete DMEM/10% FBS. Dilute in parallel control conditioned medium from untransfected A20 cells and soluble GM-CSF starting from 20 ng/ml in the control conditioned medium.
10. Add into each well 50 pl (1 x 104) MPRO cells in DMEM/10% FBS and incubate for 48 hr at 37°C.
11. Pulse cells with 1 pCi [3H]thymidine (appendix 3d).
12. Harvest cells or store plates at -20°C overnight.
13. Measure thymidine incorporation on a P scintillation counter (unit3.12 and appendix
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