In culturing L6 cells, using DMEM containing glucose with 10 % FBS. The cells would be seeded into 96 well plates and left to confluence and these would then be differentiated in DMEM with 2 % FBS for 5 days. Before tests, the medium will be replaced by RPMI1640 (2 g/L glucose) supplemented with 0.2% BSA. The medium will be removed after 2 h. My questions are:
1) How to source for this particular RPMI 1640 with this glucose strenght.
2) Hpw do I supplement the medium with 0.2 % BSA (How am I to prepare this?)
3) What should I use to remove the medium after 2 h?
Dear Lakunle,
1) 2g/L glucose should be kind of standard formulation. If not, get lower strength and add your own, then filter sterilize.
2) 0.2% BSA means 2g/L. Proceed as above. Only makes sense when there is no serum present, of course (which will bring lots of own BSA)
3) I'd use a small pipet tip attached to a vacuum source. If your cells can stand it, just flip the medium out like when you'd wash an ELISA plate. If it is a short term experiment, you may do this directly at the sink, as strong sterility is not required anymore.
And wash the plate once or twice with the new medium in order to remove traces of the old one. If medium should be too expensive, use PBS, ideally with an appropriate concentration of glucose.
RPMI-1640 standard formulation indeed has 2g/L of glucose. Just check the full formulation of the medium before purchasing it. Thermo Fisher has some RPMI-1640 with this glucose concentration.
Don´t forget to use mask while weighing the BSA, since it is toxic when in contact with your airway.
https://www.researchgate.net/.../How_to_prepare_02_Bovine...
https://www.researchgate.net/.../How_to_prepare_02_Bovine...
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