Before you start
- Titrate every antibody on your cells before the experiment.
- Plan compensation controls (single-stained cells or beads) and FMO controls for dim or continuous markers.
Reagents and materials
Totals are for 12 samples (+10% extra). Per-sample amounts are in the middle column.
| Reagent | Per sample | Total |
|---|---|---|
| PBS, protein-free (for fixable viability dye) | 0.4 mL | 0.4 mL |
| Fixable viability dye working solution (e.g. 1:1000 in PBS) | 100 µL | 100 µL |
| Fc block (anti-CD16/32 mouse or human Fc block), diluted | 50 µL | 50 µL |
| Antibody cocktail in FACS buffer (titrated) | 50 µL | 50 µL |
| FACS buffer (washes and resuspension) | 1 mL | 1 mL |
| Fixation buffer, 1–2% PFA (optional) | 100 µL | 100 µL |
Buffers and recipes
FACS buffer per 500 mL
| PBS (Ca²⁺/Mg²⁺-free) | 490 mL |
| Heat-inactivated FBS (2%) | 10 mL |
| 0.5 M EDTA, pH 8.0 (1 mM final) | 1 mL |
Filter (0.22 µm) and keep at 4 °C. 0.5% BSA can replace FBS. Sodium azide (0.05–0.1%) is optional for staining only — never for cells you will culture.
Equipment
- 96-well V- or U-bottom plate or FACS tubes
- Plate centrifuge
- Multichannel pipette
- Flow cytometer
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Plate the cells
Add 0.5–2 × 10⁶ cells per well. Centrifuge at 400 × g for 3–5 min at 4 °C and flick off the supernatant.
Wash in protein-free PBS
Wash once with 200 µL PBS (fixable viability dyes bind protein in the buffer).
Viability dye
Resuspend in 100 µL viability dye working solution and incubate 15 min at 4 °C or room temperature, protected from light.
Wash
Add 150 µL FACS buffer, centrifuge and discard.
Fc block
Resuspend in 50 µL Fc block and incubate 10 min at 4 °C.
Antibody cocktail
Add 50 µL 2× antibody cocktail (do not wash out the block) and incubate 20–30 min at 4 °C in the dark.
Wash twice
Wash twice with 200 µL FACS buffer.
Fix (optional)
Resuspend in 100 µL 1–2% PFA for 15–20 min at 4 °C, then wash. Skip if you will sort live cells.
Acquire
Resuspend in 200 µL FACS buffer, filter if clumpy and acquire. Record enough events for your rarest population.
Critical points
- Use Brilliant Stain Buffer when two or more Brilliant Violet/Ultraviolet dyes are in one panel.
- Tandem dyes degrade with light, heat and fixation — store dark, keep cold and use the same lot for compensation.
- Controls: unstained, single stains for compensation, FMOs, and a viability gate.
Troubleshooting
| Problem | What to check or change |
|---|---|
| High background | Re-titrate antibodies, include Fc block, add a viability gate, wash more. |
| Weak signal | Check antibody titre and storage; choose a brighter fluorochrome for a dim antigen. |
| Compensation looks wrong | Single stains must be at least as bright as the sample and use the identical fluorochrome lot. |
Safety
PFA is toxic: handle in a fume hood or ventilated area and collect waste.
Further reading
- Cossarizza A et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol (third edition, 2021).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.