Tamoxifen is first converted by the liver in mice to 4-hydroxy-tamoxifen, which is the active form for Cre-ERT2 to translocate into the nucleus. Therefore, in vitro you need to treat the cells with 4-OH-tamoxifen (https://www.sigmaaldrich.com/US/en/product/sigma/h7904), which you can then titrate with different concentrations. But if you transfer the cells into mice first, then you can give tamoxifen to the mouse and the mouse will make 4-OH tamoxifen for the transferred cells.

Zhixin Jing thanks for your answer, the point you answered is very important to me. sorry I didn't explain well in my question. actually i add 4-OH-tamoxifen to treat cells in vitro. and the brainbow system can work (after 2uM 4-OH-TAM treat 48h, I found color change). but with very lower effecient.

Xuelian Lei There are some possibilities that you can check:

1. The expression level of your Cre. (You can use a anti-Cre antibody to stain intracellular Cre of your cells by flow cytometry)

2. The sensitivity of your Cre to your allele. (You can use a cell that express a widely used CreERT2 as a positive control such as Rosa26-CreERT2 and culture in the same condition to compare)

3. The toxicity of the drug that if other cells are dying.

Hope this is helpful.

Zhixin

Zhixin Jing thank you so much for your suggestion. I tried add cre-recombinase and color changed.