Before you start
- Prepare Fix/Perm working solution and 1× perm buffer fresh.
- Finish viability and surface staining first.
Reagents and materials
Totals are for 12 samples (+10% extra). Per-sample amounts are in the middle column.
| Reagent | Per sample | Total |
|---|---|---|
| Fix/Perm working solution (1 part concentrate : 3 parts diluent) | 200 µL | 200 µL |
| 1× permeabilisation buffer | 0.6 mL | 0.6 mL |
| Intranuclear antibody cocktail in 1× perm buffer | 50 µL | 50 µL |
| FACS buffer | 0.4 mL | 0.4 mL |
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 plate
- Plate centrifuge
- Flow cytometer
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Surface stain
Perform viability dye and surface staining; wash once in FACS buffer.
Fix and permeabilise
Resuspend in 200 µL Fix/Perm working solution and incubate 30–60 min at 4 °C (or room temperature) in the dark.
Wash
Add 200 µL 1× perm buffer, centrifuge at 500 × g for 5 min (fixed cells pellet less well) and repeat.
Stain intranuclear targets
Resuspend in 50 µL antibody cocktail in perm buffer; incubate 30–60 min at room temperature in the dark.
Wash and acquire
Wash twice in perm buffer, resuspend in FACS buffer and acquire within 24 h.
Critical points
- This buffer quenches GFP/YFP from reporter mice; pre-fix in 2% PFA for 15–30 min or use a reporter-compatible kit.
- Increase centrifugation speed for fixed cells to avoid losses.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Dim FoxP3 | Check the clone, lengthen intranuclear staining or stain overnight at 4 °C. |
| Reporter signal lost | Pre-fix with PFA before the transcription-factor buffer. |
| Cell loss | Spin at 500–600 × g after fixation. |
Safety
Fixation buffers contain formaldehyde: ventilated area and appropriate waste disposal.
Further reading
- Manufacturer instructions for the transcription factor buffer set you use.
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.