problems with microglial culture - (Apr/01/2014 )

Hi everyone,

I have some problems with mouse microglial culture during the past several months, the cells seemed to be contaminated, but we did not find the evidence of bacteria or fungi contamination. I attached some pictures of the cells, could anyone give me some advice? I make the mixed glial culture from P1 mouse pups, and these picture were taken at D10 in culture.

Thanks!!!!!!!!! 

So what is the actual problem - lack of growth, death, rate of growth?  I can't see any evidence of contamination in your photos, but this is because of the low resolution/poor contrast - can you re-take them with white light and using phase contrast or DIC?

Other useful bits of info would be - what you are culturing them in? for how long? supplements? split ratios?

bob1 on Tue Apr 1 19:55:02 2014 said:

So what is the actual problem - lack of growth, death, rate of growth?  I can't see any evidence of contamination in your photos, but this is because of the low resolution/poor contrast - can you re-take them with white light and using phase contrast or DIC?

 

Other useful bits of info would be - what you are culturing them in? for how long? supplements? split ratios?

Thanks for your reply,

So I made the mixed glial culture and plated them in 175cm2 flask, with DMEM/high glucose, 10%FBS, 1%Antibiotics, and OPI. After 7 days I changed the medium, then I took these picture on D10. Yesterday I used CD11b beads to sort the microglia, then I saw big roundish cells, and they are GFAP positive, but they don't seems like astrocytes....

True - astrocytes should be star like. Are you sure that they are attaching to the surface properly? 

I used to work occasionally with a glioma line or two and they were very very sensitive to confluency - too confluent and they would stop growing and enter senescence - could that be the problem here?