Cells are healthy when used routinely but once freeze downs are made (FBS + 10%DMSO) and stored in -80 C their revival is quite difficult and majority cells are dead.
I am using RPMI-1640 media with 10% FBS for their revival.
How were your cells frozen? How long have the cells been at -80°C? This is not cold enough for long term storage (more then 3-6 mo); you should transfer the cells to liquid nitrogen. What is your thawing protocol?
You should thaw quickly, dilute with 10X (or more) complete media, then spin down to get rid of residual DMSO.
What is your cell density? This is *crucial* for THP-1. If just splitting, I would recommend 1e5/mL, but when thawing you may want to start at 2e5/mL or even 4e5/mL, especially if you are having viability issues.
Dear Kuldeep,
I agree with Peter, leaving the cell in -80 °C is not suitable for long term storage - the cells loses viability. You should think about storing in liquid nitrogen in future batches.
Nevertheless, if the cells are still viable (which they may not be), you can proceed like the following:
1. Add the content of the cryovial in a 15 mL tube with at least 8 mL of complete medium;
2. Centrifuge the tube at appropriate speed for your cells (such as 1 500 rpm for 2 min);
3. Remove as much supernatant as you can and resuspend the pellet in 1 mL of complete media without the formation of bubbles or much turbulance;
4. Add the content dropwise in a cell dish: try to select a small dish, as your cells might grow in a confluence-dependent manner (such as a 35 mm petri dish) - this may be important if you have many dead cells as well; or a low cell concentration when you've frozen them. Homogenize the cells in the plate;
5. After 24 h, change the medium to clean the dish of dead cells: the presence of dead cells inhibit the growth of viable ones.
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