1. Aspirate the medium, wash with 2mL DPBS twice
2. Add mL Trypsin or 0.5mM EDTA to the dishes and incubate to lift the cells
A. for iPSCs lift the cells as single cells
3. Add 2ml complete medium to stop trypsinization, and pipette up and down to collect all cells
3. Transfer all cell suspension into a 15ml conical tube, spin down to get the cell pellet 200g for 4min
4. Resuspend each cell pellet in 1ml sorting medium (Add 2% (vol/vol) KnockOut serum replacement to 50 ml of DPBS. Can be stored at 4 °C for 6 weeks.)
To make 50 mL add 1mL of KnockOut serum into 49mL DPBS
5. Prime the cell strainer with 2mL of sorting medium making sure to cover the entire mesh.
6. Discard the sorting medium in the 50mL tube
7. Apply each cell suspension to the center of a cell strainer (pushing through with pipette where necessary, and – with a new tip – pulling off strained cell suspension stuck to the bottom of filter).
8. After straining the cell suspension, add about 50µL of sorting medium to wash the strainer for any left-over cells.
9. Aliquot cell suspension into sorting tubes and put it on ice.
10. Add DAPI (diluted 1:10,000 to make final concentration at 0.1ug/ml) to the strained cell suspension. This helps to distinguish live from dead cells.