Hi, Does anyone know how to isolate the hematopoietic stem cells from rat bone marrow ?
Hello
You can follow Smajilagić method.
Briefly, after animals sacrificed via chloroform anaesthesia. Both sites, femora and tibia were aseptically removed after sacrifice under chloroform anesthesia. The cancellous bone was removed from the femur and tibia, and washed 3-5 times with 1x PBS. Thereafter, the bone marrows were flushed out using α-MEM culture medium, supplemented with 10% (v/v) FBS, freshly prepared AA (50 μg/ml), 50 IU L-1 penicillin, 2mM L-glutamine, 50 μg mL-1 streptomycin, 0.3 μg mL-1 fungizone, 50 μg mL-1 gentamycin sulphate, 10 mM β-GP, 10-8 M dexamethasone.
The released cells collected in to 75 cm2 culture flasks with 15 ml α-MEM culture medium.
The cells cultures were maintained at 37° C in a humidified atmosphere of 95% air and 5% CO2. Cells were allowed to attach for 4-5 days, when after the non-adherent cell population was removed and the culture medium was replaced with fresh culture α-MEM medium. The culture medium was changed twice a week.
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Article Rat bone marrow stem cells isolation and culture as a bone f...
Good Luck
after scarifying the mice , you can collect the BM from the thigh by cutting the peripheries of the thigh by a scissor , then inject the media (e.g. MEM) into the bone to push the BM into a flask contain the media , after that you should make a centrifugation to discard the lipid then take the precipitant (cell pellets) and overlying it on lymphocyte separation fluid (Ficoll) and centrifuge it at 400 g to take the buffy coat (cloudy layer)
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