After performing density-gradient separation, I am plating the obtained PBMCs with the intention to look at monocytes which adhere to the surface of the multi-well plate.
A lot of protocols I have looked at use DMEM/RPMI media. I use complete DMEM medium and am seeing adherence of monocytes but also see significant numbers of lymphocytes even after washing 3-5 times. My incubation time is 3 hours at 37 deg. C and I use warmed up PBS for washing.
I am wondering if one medium is preferred over the other and if so then why ?
It would help greatly if you all could share your experience in this matter.
~Ani
Hi Ani,
B cells will contaminate, they have adhesion molecules that allow them to stick to polystyrene and nylon wool. For me and human cells, to reduce contamination i used rpmi1640 10% for 3 hr at 37 w/ co2 using t flasks. I would the pour out the supwrnatant and wash 3x with cold pbs -/- ( without calcium or magnesium), using a pipette to wash the adherent surface agresdively.
Lora Benoit Thank you so much for sharing. Couple of questions:
1) When you say "10%", do you mean addition of 10% FBS ?
2) I was using PBS (-/-) at room temperature for washing. Could you explain to me why it needs to be cold ? I have read about a few protocols that suggest using cold buffer but never quite understood the reason for this.
incubating PBMCs in serum-free medium for 4-6 hr will be better to avoid such contamination. Sometimes, people keep PBMCs for overnight incubation, that may lead to the contamination of lymphocytes.
Hi Ani,
yes, 10% FBS. I'm not sure about the cold buffer reason. When I used to do this, the cells were plated in a T-65 flask and then I was aggressive with the washing step using the cold buffer.