Before you start
- Pre-chill buffer and the centrifuge to 4 °C.
- Label one tube per tissue and keep everything on ice.
Reagents and materials
Totals are for 4 spleens (+10% extra). Per-spleen amounts are in the middle column.
| Reagent | Per spleen | Total |
|---|---|---|
| FACS buffer or cold cRPMIcollection, rinsing and washes | 25 mL | 25 mL |
| ACK lysis buffer (spleen only) | 2 mL | 2 mL |
| 70 µm cell strainerone to mash, one to re-filter | 2 each | 2 each |
| 3 mL syringe plunger | 1 each | 1 each |
| 0.4% trypan bluefor counting | 10 µL | 10 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.
ACK red-cell lysis buffer per 1 L
| NH₄Cl (150 mM) | 8.02 g |
| KHCO₃ (10 mM) | 1.0 g |
| Na₂EDTA·2H₂O (0.1 mM) | 37.2 mg |
| Ultrapure water | to 1 L |
Adjust to pH 7.2–7.4, filter-sterilise and store at room temperature.
Equipment
- Dissection tools, 70% ethanol
- 6-well plate or Petri dishes on ice
- 15 and 50 mL tubes
- Refrigerated centrifuge
- Hemocytometer or automated counter
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Harvest the organs
Euthanise the mouse according to your approved animal protocol, spray with 70% ethanol, and collect the spleen and the lymph nodes you need (inguinal, axillary, brachial, cervical, mesenteric) into cold buffer on ice.
Mash through a strainer
Place a 70 µm strainer on a 50 mL tube, add the tissue with a little buffer and mash it gently with the flat end of a syringe plunger. Rinse the strainer with 10 mL buffer.
Pellet the cells
Centrifuge at 350 × g for 5 min at 4 °C and discard the supernatant.
Lyse red blood cells (spleen)
Resuspend the spleen pellet in 2 mL ACK buffer and incubate 2–3 min at room temperature. Lymph nodes usually do not need lysis.
Stop the lysis
Add 10 mL buffer, centrifuge at 350 × g for 5 min at 4 °C and discard the supernatant.
Re-filter and resuspend
Resuspend in 5–10 mL buffer and pass through a fresh 70 µm strainer to remove clumps.
Count
Mix 10 µL cells with 10 µL 0.4% trypan blue and count live (unstained) cells. Adjust to the density your next step needs.
Critical points
- Keep cells cold and work quickly; viability falls during long handling.
- Do not over-lyse: more than ~5 min in ACK damages leukocytes.
- Typical yield from an adult mouse spleen is in the order of 5–10 × 10⁷ cells after lysis; a lymph node gives a few million.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low viability | Shorten handling, keep on ice, avoid harsh mashing; add DNase I (20–50 µg/mL) if clumps appear. |
| Red pellet after lysis | Repeat a short (1 min) ACK step or use fresher ACK buffer. |
| Clumps in the suspension | Re-filter; include 1–2 mM EDTA in the buffer; avoid vortexing. |
Safety
Work under your approved animal protocol. Dispose of sharps and carcasses as required.
Further reading
- Current Protocols in Immunology, unit on isolation of mouse lymphoid cells.
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.