Before you start
- Prepare a single-cell suspension and count it.
- Check your kit for exact volumes — they differ between suppliers.
Reagents and materials
Totals are for 2 spleens (+10% extra). Per-spleen amounts are in the middle column.
| Reagent | Per spleen | Total |
|---|---|---|
| Isolation buffer (PBS + 2% FBS + 1 mM EDTA) | 10 mL | 10 mL |
| Biotin antibody cocktail (per kit)per ~10⁸ cells | 1 kit dose | 1 kit dose |
| Streptavidin magnetic beads (per kit)per ~10⁸ cells | 1 kit dose | 1 kit dose |
Buffers and recipes
Isolation buffer per 500 mL
| PBS (Ca²⁺/Mg²⁺-free) | 489 mL |
| FBS (2%) | 10 mL |
| 0.5 M EDTA (1 mM) | 1 mL |
Degas if using columns; keep cold.
Equipment
- Magnet for 5 mL tubes (or columns and separator)
- 5 mL polystyrene tubes
- 30–40 µm filter
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Adjust density
Resuspend cells at the density in your kit (often 1 × 10⁸ cells/mL) in cold isolation buffer and filter.
Antibody cocktail
Add the biotinylated antibody cocktail (antibodies against non-CD4 cells), mix and incubate on ice or at room temperature as the kit says (commonly 10 min).
Magnetic beads
Add streptavidin beads, mix and incubate (commonly 5–10 min).
Separate
Top up with buffer, place in the magnet for 2.5–5 min and pour off or collect the untouched (enriched) cells.
Optional second round
For higher purity, repeat the magnet step on the poured-off fraction.
Check purity
Count and stain a small aliquot for CD4 (and CD62L/CD44 for naive cells). Expect more than 90% purity.
Critical points
- Accurate counts matter: too many cells per bead dose lowers purity.
- Keep the suspension free of clumps; filter before the magnet.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low purity | Reduce cell number per dose, add a second magnet round, check bead mixing. |
| Low yield | Do not leave tubes in the magnet longer than stated; collect wash fractions. |
Safety
Standard BSL-1/2 practice for primary cells.
Further reading
- Kit instructions (EasySep, MojoSort, MACS or similar).
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.