I need to know what concentration of cycloheximide can be used in HeLa Cells without killing the cells. According to Santa Cruz Biotech that sells this compound, IC50 value of CHX is 532nM for Hela Cells. I have also read that this value is cell line specific. I have come across papers that have used it at 10-30ug/ml for this cell line. But I also calculated that 532nM corresponds to 0.14ug/ml conc of CHX. So they are using it at very high levels. I want to avoid doing a cytotoxic assay, so I would need valid references for supporting the use of a specific concentration of CHX. I plan to use it for 3-4 hours in HeLa Cells having overexpressed luciferase and want to inhibit any protein synthesis after i give the cells Heat Shock. Also, I think I have to add CHX after Heat Shock treatment, but please confirm if it is otherwise.
For all practical purpose that you indicated, a range between 5-15 microgram/ml is predicted to inhibit protein synthesis. Because the stability of CHX decreases above pH 7.0, one needs to make sure that the pH of the cell culture medium stays below 7.0. If the goal is to inhibit protein synthesis following a heat shock to cells, cells need to be treated before the shock (- 15 min) to inhibit protein synthesis following the shock (as a control, no CHX treatment before heat shock).
1-20 ug/ml for 4-24h, ideally do a pilot Western blot expriment to see if your favorite protein marker disappears as a result of the treatment. Ubiquitin is a good sensitive marker for Cycloheximide treatment.
In addition to above comments, I wish to add that in our recent publication we used 10 micromolar cycloheximide for 4 h to inhibit protien translation in primary cells (bone marrow derived macrophages). This efficiently inhibited protien synthesis. Further, if you want to look at your m RNA on polysomes cycloheximide is used at 100 microgram per mL in PBS for 10 minutes on ice at the time of harvest.
Feel free to check our paper here doi: 10.1074/jbc.M117.785014
Hi Bhaskar, could you please let me know what concentration of cycloheximide did you use in HeLa Cells..
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