Hello,
I am having trouble obtaining large amounts of macrophages in culture. I obtain them from SD rats bone marrow and sort them before culture. I let the sorted monocytes sit for 7 days (adding some fresh medium on day 3), and when they are close to confluent I split them with the intention of increasing my population.
After I replate them, they are not sick, they look healthy and attach again, but they do not become confluent again. Is that an intrinsic property of the cells or am I doing something wrong? Shall I change my detaching method?
Culture medium: RPMI-1640 + 1:1000 Gentamycin + 10% FBS + 50ng/ml M-CSF
Detachment method: Rinse twice with HBSS at RT, Incubate at 37°C with 20mM EDTA-HBSS for 10-15 min, tap on the side and harvest. They come off fairly well and show 70-80% live count.
Thank you!!
Hi
What is your seeding density for subcultures?. If it is low u can increase the density.
You can also try to increase FBS concentration for fresh subcultures.
One should only use them up to 5 passages as their properties might
change over time
What culture dishes are you using? The use of tissue-culture treated dishes can accelerate their differentiation and slow proliferation. If you untreated 100 mm petri dishes you may also improve your yield.
Hi Mamatha,
Thank you for your answer. I seed them at about 1-1.6 x 10^5 cells / mm squared. So if I sort about 10 million cells I will plate them on a 100 mm dish. I had noticed that they proliferate and survive better at higher densities.
I have never been able to reach more than 2 passages yet... How much do you increase the serum? I would like to try that.
Also, do you have any recommendation for macrophage freezing medium?
Thank you Jodi. I use untreated petri dishes, just normal plastic at different sizes.