I'm trying to perform the MLA. I'd like to have some suggestions about 3 issues:
- does anyone use a test to verify cells purification with methotrexate?
- which are the concentrations of media supplements (thymidine, hypoxanthine, glycine and methotrexate)?
- how do you perform cell density adjustement during the phenotypic expressione period?
Thanks,
Angela
- I never use any test to verify the mutant cleansing and I don't know if such test exists.
- I prepare THMG 100X: 1.2 mg/ml Thymidine and 2 mg/ml Hypoxanthine both in HCl 0.1 M; Glycine 3 mg/ml in water; Methotrexate 1 mg/ml in NaOH 0.1M then dilute 25-fold in water. Finally I join same volume of each solutions to obtain THMG 100X.
- 24 hours after exposure I prepare 2 F75 with 4 X 106 cells each. The day after, if some culture has less than 3 cellular divisions in 48 h I do not plate it in TFT medium. I prepare again 2 flasks with 4 X 106 cells/F75 and delay the mutant selection to 3rd day but only for such cultures.
Bye
Duccio
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