Hello, since I have no experience in Tet-on system in drosophila S2 cell culture, I need some technical help...
- Should I add P/S into the cell culture media?
- What's the doxycycline concentration commonly used for S2 cells? (I have seen people use 100ng/ml - 1 ug/ml)
- Should I add doxycycline at least 24 hours after the transfection?
- ANY tips/good suggestions for doing this type of experiments?
Thank you all in advance!!!
Follow, such an intresting question.
In my simple experience in tissue culture, yes should add antibiotics with media culture to prevent microbial contamination.
We have been using the Tetracycline-regulated expression system for many years. We optimized the ‘Tet-on’ system to obtain a tightly regulated Tet-inducible expression system, which has been tested in several different experimental studies, mostly using mammalian cells (Arthritis & Rheumatism, 2005, Vol. 52(9), p 2906 – 2916; Arthritis Research and Therapy. Jan. 2007 9(1) R7; Yousaf & Gould, The Tetracycline responsive system, Methods Mol Biol 2017, 1651, 159-172, https://www.ncbi.nlm.nih.gov/pubmed/28801906). Following the transfection procedure, I normally culture cells in complete medium for 16 – 20 hours and then change the medium. Complete medium containing doxycycline (0.0001 - 1 μg/ml) is added to the transfected cells and cultures continued for 16 – 24 hours, as appropriate for the study. In one study, the rat neuroblastoma B104 cells cultured in medium containing 1 μg/ml of doxycycline for up to 4 days were analysed daily by FACS for expression of the target protein (BBA - Molecular Cell Research, 2011, 1813(8): 1428-1437).
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