To prepare a gelatin coated 24 well plate add 200 microliters of the 0.2%gelatin solution to each well. Gently shake the plate to evenly spread the solution and allow the wells to coat at room temperature. After 15 minutes, using a vacuum aspirator carefully remove any leftover gelatin from the wells.
Add 0.5 milliliters of freshly prepared NBIL media to each well. And place the plate in a tissue culture incubator to prewarm. Aliquot 30 milliliters of wash media into a 50 milliliter conical tube.
Aspirate the media from the tissue culture dish of mESCs and add required amount of cell detachment medium. After three to five minutes, pipette up and down 15 to 20 times using a P1, 000 pipette. Observe the cells under a microscope.
To ensure a single cell suspension transfer the cell suspension into a 50 milliliter tube containing wash media. Centrifuge the suspension at 300G for five minutes at room temperature and aspirate the wash media. Resuspend the pellet in approximately 300 microliters of wash media.
