biomedical master student here , i am dealing with mice BMDM and would like some help , i seed 0.5 x 10^6 cells/per well in a 6 well plate but when i extract the RNA i get a very low yeild , i suspect that the problem is with the low cell number but i am not sure .
Also , does anyone know a protocol for freezing mice BMDM , i lloked around but didn't find a comprehensive one , so i tried freezing before lysing and another sample i froze just before plating , but i am looking for someone who has experince with this for guidance
thanks in advance
Hello Ibrahim
You can use 2 million cells per well in a 6-well plate. If you wish to get a higher yield of RNA you can pool two wells.
Regarding the freezing medium for BMDM you can use 90%FBS and 10% DMSO. Freeze about 2 million cells per ml per cryovial.
Regards.
Malcolm Nobre
thank you
would you suggest I freeze them before lysing , or before plating , i am trying the 2 methodes , but looking for more suggestion to decide which would work better .
Yes
your problem is definitely low cell number
How are you culturing your cells and extracting RNA
with that info I might be able to help
Laurence
Laurence Stuart Dawkins-Hall
After i isolate them by flushing from the femur and tibia , i count cells and plate 10 million into 10 cm tissue culture plates with 7 ml of medium+ MCSF , medium is DMEM/F-12[HAM] , and then 3 days later i supplement with another 3 ml , and 2 days after that , I scrape and plate into 6 well plates 500k into each well .
It sounds like you are stuck with that cell no. Which could be a problem
It is then about how you extract the RNA
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