I have been applying a double thymidin block to my NIH3T3 cells to synchronize them. The aim is to fix them in mitosis and perform some immunocytochemical stainings. Although I see a good amount of rounded cells before fixation, after staining procedure, I seem to have lost all of them. I seeded cells in 24well cell culture dishes and perform the whole staining in the dish. I was very careful not to apply much force during the washing and changing of liquids steps. However, no mitotic cell was left.
Dear Franziska ,
The double thymidine block synchronizes cells at the G1/S border of the cell cycle. After removing the second thymidine block, you must wait for a time equivalent to the duration of phases S and G2 for maximal % of mitosis. I can recomend you at this moment to add fixator directly to the cell culture medium.
Best regards,
Rustem Uzbekov
PS You can also see a detailed description of the different methods of cell synchronization in my review.
Article Analysis of the Cell Cycle and a Method Employing Synchroniz...
Rounded cells in mitosis have minimal adhesion to their substrate. So you need to try various way to increase that adhesive ability. You can buy matrix-coated dishes, coat the wells yourself or use other non-matrix means like polylysine. In addition, try fixing in PFA at room temperature since it will fix at a faster rate. Also, try to due the staining procedure in a single day to ensure that there is not an opportunity for the cells to be altered while in a buffer wash for too long.
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