Standard Protocol for Sorting Cultured Cells

While planning for sorting

1. Make a rough estimate of the cell diameter under microscope (while floating NOT when the cells are attached to the plate).
2. Let the flow core know the cell diameter info before you book the sorter, this is a crucial information for setting the sorter properly.
3. Consult the core to choose best collection device for the experiment.

Pre-sort

1. Wash your cells ~70% confluency with cold PBS twice.
2. Detach cell from the plate as gentle as possible. Harsh detachment like treating cells with Trypsin for too long will create false positive dead cells.

How to bring cells to the core

Ideal cell concentration to bring as follows Cell diameter under 15 micron (15 to 20 million/ml), Cell diameter between 15 to 17 micron (10 to 15 million/ml), Cell diameter between 17 to 22 micron (5 to 10 million/ml).

Make sure to use 0.2% BSA containing HEBS for cell resuspension. HEBS will keep cells alive for much longer period compared to PBS or any cell culture media. Bring cells in ice not in RT.

How to capture sorted cells

You can collect cells in various types of plate, 15 or 50 ml tubes, FACS tube or Eppendorf. Please check the following link to learn how to coat tubes for best results.

https://medicine.ecu.edu/flow-core/sort-capture-tube-preparation-protocol/

For sorting

1. Add 1 to 2 µg/ml DAPI in the sample to detect dead cells, you can add just before the sort
2. Bring additional HEBS buffer, in case sample needs to be diluted
3. You may bring 30 to 70 micron filter caps to filter out cell aggregates if needed.
4. Bring some unstained cell (for transfected cells this would be parent/untransfected cells) as reference

Post sort

1. Collect cells in any buffer, media as you wish.
2. Add minimum 2X antibiotic in the culture media. Wait for 24 hr or cells to be attached, wash and replace the media with fresh 2X antibiotic containing media for 24 hr. Then use regular media. You can also use 50 µg/ml of Gentamycin for additional safety.