Question

How would you prepare adherence cells (tissue culture) in a flow cytometry?

Answer 1

Preparation of adherent tissue culture cell lines:

This method provides a general procedure for use with adherent tissue culture cells

1. Prepare PBS-Phosphate Buffered Saline - containing 1% BSA(Bovine Serum Albumin)
2. Harvest cells by enzymatic release using 1x Accutase solution or 0.25% trypsin, followed by quenching with media containing serum. (Note: epitopes may be cleaved when using the enzymatic digestion method. Cells can also be harvested by gently scraping them into culture media)
   1. Remove the culture medium and eliminate residual serum by rinsing cell monolayers with sterile, room temperature PBS.
   2. Slowly add 1x Accutase solution or 0.25% Trypsin to cover the cell monolayer.
   3. Incubate at 37^oC for up to 10 minutes.
   4. After incubation gently tap the flask and the cells will detach and slide off in one sheet to the bottom of the flask.
   5. Add growth medium and re-suspend the cells by gently pipetting.
3. Centrifuge at 300-400 g for 5 min.
4. Discard supernatant and resuspend pellet in fresh, room temperature PBS/BSA to wash off any remaining cell debris and proteins.
5. Centrifuge at 300-400 g for 5 minutes at room temperature.
6. Discard supernatant and resuspend pellet in an appropriate amount of room temperature PBS/BSA.
7. Count cells using a hemocytometer or an automated cell counter such as the TC20^TM Automated Cell counter.
8. Once counted dilute the cells with cold (4^oC) PBS/BSA to a minimum concentration of 1 x 10⁷ cells/ml.