Protocol: Preparation of Tissue Culture Cell Lines for Flow Cytometry

Reagents

• Phosphate buffered saline (catalog BUF036A) containing 1% bovine serum albumin (PBS/BSA)
• 1x Accutase Solution or 0.25% trypsin
• Blocking reagent: Mouse Seroblock FcR (BUF041) or Human Seroblock (#BUF070)

Preparation of Cells Stored in Liquid Nitrogen 

1. Carefully remove cells from liquid nitrogen storage.
2. Thaw cells rapidly in a 37°C water bath.
3. Resuspend cells in 10 ml cold PBS/BSA buffer and transfer them to a 15 ml conical centrifuge tube.
4. Centrifuge at 300–400 g for 5 min at 4°C.
5. Discard supernatant and resuspend pellet in cold (4°C) PBS/BSA and adjust cell concentration to
1 x 10⁷ cells/ml.

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

Preparation of Tissue Culture Cell Lines in Suspension

1. Decant cells from tissue culture flask into 15 ml conical centrifuge tube(s).
2. Centrifuge at 300–400 g for 5 min at room temperature (RT).
3. Discard supernatant and resuspend pellet in 10 ml of RT PBS/BSA.
4. Centrifuge at 300–400 g for 5 min at RT.
5. Discard supernatant and resuspend in cold (4°C) PBS/BSA and adjust cell concentration to
1 x 10⁷ cells/ml.

Preparation of Adherent Tissue Culture Cell Lines

1. Harvest cells by enzymatic release using 1x Accutase Solution or 0.25% trypsin, then quench 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.

a.  Remove the culture medium and eliminate residual serum by rinsing cell monolayers with sterile,
RT PBS.
b.  Slowly add 1x Accutase Solution or 0.25% trypsin to cover the cell monolayer.
c.  Incubate at 37°C for up to 10 min.
d.  After incubation, gently tap the flask and the cells will detach.
e.  Add growth media and resuspend the cells by gently pipetting.

2. Transfer cells into a 15 ml or 50 ml conical centrifuge tube and centrifuge at 300–400 g for 5 min.
3. Discard supernatant and resuspend pellet in fresh, RT PBS/BSA to wash off any remaining cell debris and proteins.
4. Centrifuge at 300–400 g for 5 min at RT.
5. Discard supernatant and resuspend pellet in cold (4°C) PBS/BSA and adjust cell concentration to 1 x 10⁷ cells/ml.

Notes

• Ensure you prepare enough cells for your experiment. Typically 10⁶ cells are used for each flow cytometry sample.
• To ensure that cells are in a single cell suspension and to  remove debris, pass sample through a 40 µm cell strainer just prior to counting. 
• Count cells using a hemocytometer or an automated cell counter such as the Bio-Rad TC20 Automated Cell Counter (1450102).
• It is best practice to block Fc receptors to avoid nonspecific binding. To block human cells, we recommend using Bio-Rad’s Human Seroblock (BUF070), and to block mouse cells, we recommend Bio-Rad’s Mouse Seroblock FcR (BUF041).