I am having problem of mouse bone marrow stromal cells primary culture. First 2-3 days I see cells very healthy but then slowly hazzy and dying. I use alpha MEM media with 10% FBS and 1x glutamine and 1x antibiotics. I even tried if there is contamination in media or during isolation of bone maarow, but could not find that. So, please give me best idea for mouse BMSCs culture.
Try following tips...and let me know
Collect marrow from young animals.
Do not over incubate the collected buffycoat within RBC lysis buffer.
As concentration will be less, try to do primary culture in 24 well plate,
Initially use less media and later top up after cell attachment.
Is recommended to use the DMEM low glucose and serum possible to increase the level to 15% and reservists from alcohol during sterilization
You can use one of the following :
Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG)
supplemented with 15% heat inactivated fetal bovine serum (FBS)
penicillin/streptomycin (100 U/mL and 100 ?g/mL) .
or use , RPMI medium supplemented with 10% FCS, 100 IU/ml penicillin and 100 µg/ml streptomycin.
as mentioned Mr.ِِahmed , must be increase the level sterilization at every steps
Hi Sher,
I agree with Pratheesh all suggestions, apart from that other valuable comments also important...
My suggestion is, use DMEM-F12 medium for mouse BMSCs culture, because currently i am doing this types of experiments, i always used this medium with 10% FBS, 1% penicillin/streptomycin...
Good Luck!!!
As I am experiencing pretty good results with RPMI+10%FCS(Heat inactivated) and additionaly 1% Pen/Step to facilitate with your cell culture. I hope you will get better result with Bone marrow MSC.. Good Luck
The seeding density should be concerned for animal BMMSC culture, especially during the primary culture.
I would like to thank you all very much for giving me ideas. I got rid of the problem. Now the cells growing well. But having problem with the subculture. I am using 0.25% Trypsin with 1X EDTA, rat bone marrow stromal cells detach well in short time 2-5 mins but mice bone marrow stromal cells don not detach till 10 minutes. Is there any better ideas for proper detachment in short time????
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