Hello everyone. Now I have troubles with tumor cell culture. First I thaw cell stock and keep culture. When it become 80% confluence, I passage tumor cells. And use 1:40 dilution to keep culture. Usually I will keep 3 days. But after passage one or two times, cell growth speed seems slower. Is it because I seed cell number too low? 1:40 maybe not suitable. And how much dilution is better?
And lower density will affect cell conditon? I make lysate with lower confluence cell and that western results are different from before. Now I focus on tumor cell stress and metabolism. So each time different cell density will affect my western results?
I am really appreciate for your kindness help.
PS, I attached my cell photos,which I thought it was 80% confluence, please help me to confirm it is or not.
Hello Zhao Weiyang
4T1 is a murine mammary carcinoma cell line. Sub-cultivation ratio of 1:6 to 1:8 is recommended. You can add fresh culture medium every 2-3 days. You should not use 1:40 dilution. The seeding density is too low. Cell density affects the expression levels of multiple proteins residing in different cellular compartments. There may be a remarkable variation in the level of expression of several proteins as a function of cell confluency. Therefore, a correct and standardized cell seeding protocol is a critical factor for reproducible experimental results.
Best.
I am agree with Malcolm Nobre . Low cell concentration is the main reason. Proliferation of a cell depends on the balance between outer and inner situations. One of the most important outer signal for a cell to proliferate is cell-cell interactions. These interactions have crucial role on cellular metabolism and regulation of cell cycle. You can imagine like they need friends references to proliferate :). Therefore, dilute concentration of cells ends with senescence and decline phase unfortunatelly. Maybe you can increase FBS concentration in media to support your cells for proliferation.
1:40 is way too low in seeding density for 4T1 and most cell lines. Many cells would require autocrine or metabolites from neighboring cells to grow. When you need to grow those cells at low density, you may need to blend in some conditioned (old) medium. Also, do not allow the cells to overgrow as that would slow down the cell growth by contact-inhibition too.
I am also culturing 4T1 cells, and I use 1* 10^6 cells per T75 flask (15 ml final volume). I was wondering what the maximum number of cell passage should be ? My cell's growth seems to be slowing down a lot after passage 20.
Thanks
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