Greetings Heroes of the Scientific Vanguard!
I'm starting out with saying I'm new to this. This means that things you might take for granted, is not as obvious to me.
My problem lies with, as the topics suggest: Monocytes won't adhere to the bottom of my "Tissue culture treated" Corning flasks after Ficoll.
My procedure is as follows:
I receive a fresh buffy coat. I dilute 1:1 with PBS. Overlay 16 mL of the blood:PBS mix on 11 mL of Ficoll in 50 mL Falcon-tubes. Centrifuge them for 30 minutes at 850 g/RCF, at 20 degrees Celsius. I take the PBMC layer put it in a Falcon-tube with PBS. Centrifuge at 400G for 10 minutes two times. Then a wash at 200G for 5 minutes to remove platelets.
I then count the cells and adhere 250 million in 30mL RPMI 1640 medium with 2% human AB serum in 175 cm2 Corning "Tissue-culture treated" flasks. I let them adhere for 2 hours in the incubator. But not many cells adhere. And even more come of when I so gently, gently "wash" the flask with PBS.
I have seen, some of the times, that I have a lot of cell debris, which step is most crucial to not accidentally killing the cells?
Is there anything else in my procedure that might be the villain in all of this? Any ideas on what I could try? Adding EDTA to the PBS?
Between the washes I resuspend the cells by drawing the tubes against a grating in my bench (as to my tutors instructions). Which I realized just now might be sort of... violent.
Thanks for all and any help you can give me!