To begin, maintain HEK293T cells in culture media containing DMEM with 10%FBS in a humidified incubator. Check the plate's confluency under a microscope. When cells reach approximately 80 to 90%confluency, remove the media and wash them with 10 milliliters of DPBS.
Then, remove the DPBS from the culture dish. Treat the cells with one milliliter of 0.05%trypsin-EDTA phenol red and incubate the cells for three minutes at 37 degrees Celsius. After removing the plate from the incubator, add nine milliliters of culture media to stop the trypsin reaction, and mix by pipetting to dislodge any remaining cells.
Now, transfer the cell suspension to a 50-milliliter tube. Centrifuge at 300g for five minutes at room temperature. Once the media is removed, resuspend the cell pellets in one milliliter of fresh media.
To plate cells in a six-well plate, combine the necessary volume of cells with 13 milliliters of media in a 50-milliliter tube. Then, dispense two milliliters of the diluted cell suspension into each of the six wells and gently tap the plate on all sides to spread the cells evenly. Afterward, place the plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide overnight.
