Protocol: Preparation of Cells for Flow Cytometry

Experience challenges in harvesting cells from cell lines? Here is a protocol for efficient harvesting of cells from tissue culture.

Single cells must be suspended at a density of 10⁵-10⁷ cells/ml to keep the narrow bores of the flow cytometer and its tubing from clogging up. The concentration also influences the rate of flow sorting, which typically progresses at 2,000-20,000 cells/second. Phosphate Buffered Saline (PBS) is a common suspension buffer.

The most straight forward samples for flow cytometry are non-adherent cells from tissue cell culture. Here we describe methods for both tissue culture cell lines and adherent tissue culture cell lines.

Reagents

• PBS/BSA (PBS containing 1% Bovine Serum Albumin (BSA))
• PBS
• Accutase solution 1X
• Trypsin 0.25%

Cells stored in liquid nitrogen

1. Prepare cold PBS/BSA buffer.
2. Carefully remove cells from liquid nitrogen storage.
3. Thaw cells rapidly in a 37^oC water bath. Resuspend cells in cold PBS/BSA buffer and transfer to a 15 ml conical centrifuge tube.
4. Centrifuge at 300–400g for 5 minutes at 4^oC.
5. Discard supernatant and resuspend pellet in an appropriate amount of cold PBS/BSA buffer, such as 10⁷ cells/ml.

   Note: higher viability can be obtained by allowing the cells to recover in culture media overnight.

Tissue culture cell lines in suspension

1. Prepare PBS/BSA buffer.
2. Decant cells from tissue culture flask into 15 ml conical centrifuge tube(s).
3. Centrifuge at 300–400g for 5 minutes at RT.
4. Discard supernatant and resuspend pellet in 10 ml of PBS/BSA at RT.
5. Centrifuge at 300–400g for 5 minutes at RT.
6. Discard supernatant and resuspend to a minimum concentration of PBS/BSA, such as 1×10⁷ cells/ml in cold 4^oC PBS/BSA.

Adherent tissue culture cell lines

1. Prepare PBS/BSA buffer.
2. Harvest cell by enzymatic release using a solution containing 1x Accutase 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.)

   – Remove the culture medium and eliminate residual serum by rinsing monolayers with sterile PBS buffer at RT.
   – Slowly add 1x Accutase solution or 0.25% trypsin to cover the cell monolayer.
   – Incubate at 37^oC for up to 10 minutes.
   – After incubation gently tap the flask and the cells will detach and slide off in one sheet to the bottom of the flask.
   – Add growth medium and resuspend the cells by gently pipetting.
3. Centrifuge at 300–400g for 5 minutes at RT.
4. Discard supernatant and resuspend pellet in fresh PBS/BSA at RT in order to wash off any remaining cell debris and proteins.
5. Centrifuge at 300–400g for 5 minutes at RT.
6. Discard supernatant and resuspend pellet in an appropriate amount of PBS/BSA buffer at RT.
7. Count cells using a hemocytometer or by using an automated cell counter such as Bio-Rad’s TC20™ Automated Cell Counter.
8. Once counted dilute the cells with cold (4^oC) PBS/BSA buffer to a minimum concentration of 1×10⁷ cells/ml.