I would like to set up an in vitro culture of cells that originate from a CreERT2 transgenic mouse. Normally this system is activated in vivo with tamoxifen but I would like to induce this system in vitro. The problem is that I am not sure what is the general protocol of that? What concentration of tamoxifen can I use before it becomes toxic to the cells? How long shall I leave it in the medium to fully activate the system? I will be grateful for any suggestions.
hello, no I did not, however I have generated my own protocol and it seems to work. I use 3ug/ml of tamoxifen (dissolved in DMSO) in a culture medium. I culture my cells in a 12 well plate and use 1ml of culture medium per well. Tamoxifen treatment is not toxic to my cells but you might wanna do a test assay to find out how well your cells cope with it. Good luck!
Hello Marta,
I can offer suggestions on the first aspect of your question. Generally in order to use a drug compound in an in-vitro system, you will first of all have to establish the toxicity level it has on the cells. In other words, you should know what concentration is suitable for sustained viability . The most commonest detection assay employed is the MTT or XTT assay, where you expose the cells to graded concentrations of your drug and see how it affects their viability. You can also decide to have different time frames following drug exposure e.g you can have 24, 36, 48, 72 hrs, and read the assays after every time interval. The results can then be compared to see the effect of concentration on the viability of the cell over time, this will perhaphs give an insight into your second question; How long?.
You can check more detailed post on MTT and XTT to get up to speed on that.
Hope this helps.
Good luck!
Does anyone have specific references for exogenous Tam application with ERT2 cells?
Hi Marta and Lachlan, did you get more specific information or protocol? Thanks.
hello, no I did not, however I have generated my own protocol and it seems to work. I use 3ug/ml of tamoxifen (dissolved in DMSO) in a culture medium. I culture my cells in a 12 well plate and use 1ml of culture medium per well. Tamoxifen treatment is not toxic to my cells but you might wanna do a test assay to find out how well your cells cope with it. Good luck!
But be sure to use 4-hydroxy Tamoxifen in cell cultures as opposes to use of tamoxifen in vivo.
In my cell culture of primary keratinocytes and a carcinoma cell line, TAM (4OH, dissolved in EtOH at 10mg/ml) seems to be toxic above 0.2 ug/ml in serum-free keratinocyte medium. Cells start to die after 3h of incubation.
I produced knock-In of CRE-ER in those cells and I would like to test the CRE-ER activity through the transfection of Cre Stoplight 2.4 in the presence of TAM.
I wonder if concentrations lower than 0.2ug/ml are enough to see an effect and for how long should I incubate with TAM after transfection with Cre Stoplight 2.4.
Thank You.
Luca
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