Mouse Blood Processing to Single-Cell Suspension

Dillon Corvino

Processing of mouse peripheral blood to obtain leukocyte-enriched single-cell suspensions for flow cytometry and immunological assays.
Published

September 18, 2026

Protocol ID: MUS-BLD-PRC
Version: v1.2
Author: Dillon Corvino

Purpose

This protocol describes the processing of mouse peripheral blood collected in EDTA to generate a leukocyte-enriched single-cell suspension using RBC lysis and gentle washing. The final suspension is suitable for flow cytometry, basic immunophenotyping, and other standard immunological assays.

Critical notes (read before starting)

  • Always collect blood into tubes containing EDTA (or other approved anticoagulant) and mix immediately to prevent clotting.
  • Perform all processing steps as soon as possible after collection; avoid prolonged storage at room temperature.
  • RBC lysis should be time-limited (~5 minutes) to minimise leukocyte damage.
  • Keep samples on ice or at 4 °C after lysis and during washing to preserve viability.

Approximate timing

  • Blood collection and transfer: 2–5 min per mouse
  • Initial spin and serum removal: 5 min
  • RBC lysis and wash: 10–15 min

Total hands-on time per sample: 20–25 minutes, depending on batching.

Procedure

STEP 1 – Blood collection

  1. Prepare EDTA-containing microcentrifuge tubes (20–40 µL EDTA per tube) and label one tube per mouse.
  2. Collect blood using an insulin syringe (or other approved method, e.g. retro-orbital, submandibular, cardiac puncture as per ethics approval).
  3. Immediately dispense the blood into the EDTA tube.
  4. Flick the tube gently to mix blood with EDTA and prevent clotting.
Warning

Ensure local ethics approval covers the chosen blood collection method. Handle sharps carefully and discard immediately into an approved sharps container.

STEP 2 – Allocate sample volume and Serum removal

  1. Transfer a defined volume of well-mixed whole blood (e.g. 100–200 µL, or as required) from the EDTA tube into a new, labelled microcentrifuge tube.
    • Record the volume used per sample in your lab notebook.
  2. Centrifuge the whole blood at maximum g (bench-top microcentrifuge) for 5 minutes at room temperature or 4 °C.
  3. Carefully remove the serum:
    • Discard, or
    • Aliquot and freeze at −20 °C or −80 °C if required for serum analyses.

STEP 3 – RBC lysis

  1. Gently resuspend the remaining blood pellet in 1 mL of 1× BioLegend RBC lysis buffer.
  2. Transfer the suspension to a 15 mL Falcon tube.
  3. Gently mix by inversion or brief pipetting to ensure complete resuspension.
  4. Incubate for 5 minutes at room temperature.
Warning

Do not exceed the recommended lysis time; prolonged exposure to RBC lysis buffer can damage leukocytes and alter marker expression.

STEP 4 – Wash and final resuspension

  1. After the 5-minute lysis, top up the tube to 10 mL with FACS Buffer.
  2. Centrifuge at 400 g for 4–5 minutes at 4 °C.
  3. Carefully pour off or aspirate the supernatant.
  4. If substantial red cell contamination remains:
    • Repeat the RBC lysis step (STEP 3) once more, followed by the wash in STEP 4.
  5. Finally, resuspend the cell pellet in an appropriate volume of FACS Buffer (e.g. 0.5–1 mL) for:
    • Flow cytometry staining
    • Cell counting
    • Other downstream assays

Keep the cell suspension on ice until staining.

Buffers Required

Full composition, preparation, storage, and safety information for each buffer lives in its own linked buffer document — only a concise per-buffer summary is reproduced here, generated automatically from that document.

FACS Buffer

Component Final concentration / amount
PBS 1x to 500 mL
FCS 10 mL (2% v/v)
  • Final volume: 500 mL
  • Preparation note: Mix gently by inversion.
  • Sterilisation / filtering: 0.22 um filter if sterility required
  • Storage: 4C, 4-6 weeks
  • Caveat: No EDTA - EDTA is excluded from FACS buffer to avoid interference with staining reagents and functional assays. Use MACS Buffer (BUF-MACS-BUF) instead if EDTA is needed.

Full composition, preparation steps, storage, and safety information: BUF-FACS-BUF.

RBC Lysis Buffer (HybriMax)

Component Final concentration / amount
RBC Lysis Buffer (HybriMax), 1x ready-to-use as supplied
  • Final volume: 0.5 mL per sample (as used in MUS-LIV-DIG)
  • Preparation note: If supplied as concentrate, dilute to 1x per manufacturer instructions before use.
  • Storage: 2-8C per manufacturer instructions; do not freeze unless explicitly supported
  • Caveat: Used at 0.5 mL of 1x buffer per sample with a 7-minute room-temperature incubation in MUS-LIV-DIG - adapt incubation time if switching supplier.

Full composition, preparation steps, storage, and safety information: BUF-RBC-HYB.

ACK RBC Lysis Buffer

Component Final concentration / amount
Ammonium chloride (NH4Cl) 8.26 g/L
Potassium bicarbonate (KHCO3) 1.00 g/L
EDTA 0.037 g/L (37 mg/L)
  • Final volume: 1 L
  • Preparation note: Dissolve in sterile distilled water, then adjust to pH 7.2 with dilute HCl before sterile filtration.
  • Sterilisation / filtering: 0.22 um filter required
  • Storage: 4C; stable up to 3 months when sterile; do not freeze.

Full composition, preparation steps, storage, and safety information: BUF-ACK-LYSIS.

Materials

Reagents

Reagent Supplier / Cat# Notes
EDTA [TBD] 20–40 µL per collection tube
BioLegend RBC Lysis Buffer (1×) [TBD] 1 mL per sample; may repeat once
FACS Buffer In-house (see buffer) BUF-FACS-BUF; for washing and resuspension
PBS 1× Any As needed

Disposables

Item Notes
Microcentrifuge tubes (1.5–2 mL) 2 per mouse (EDTA collection + processing)
15 mL Falcon tubes 1 per sample
Insulin syringes / collection needles 1 per mouse (as per procedure)
Transfer pipettes For serum removal and transfers
Gloves, bench wipes, sharps container Standard PPE and safety disposables

Equipment

Equipment Notes
Bench-top microcentrifuge For initial high-g spin
Centrifuge for 15 mL tubes 400 g at 4 °C
Biosafety cabinet (Class II) Recommended for handling blood
Timer For lysis timing

Troubleshooting

Issue Possible cause Suggested solution
Sample clotted Delayed mixing with EDTA Mix immediately after blood collection; discard clotted samples
Persistent red tinge after lysis Incomplete RBC lysis Repeat lysis once; do not extend lysis time excessively
Low leukocyte recovery Pellet loss during washes Pour supernatants slowly; consider aspirating instead
Poor viability Prolonged room-temperature handling Keep samples cold; minimise processing time

Safety (brief)

  • Treat all mouse blood as potentially infectious; follow S1/S2 biosafety rules.
  • Use appropriate PPE (lab coat, gloves, eye protection) and handle sharps with care.
  • RBC lysis buffers may contain toxic components (e.g. ammonium chloride, azide); avoid contact with skin and eyes and dispose of waste according to institutional chemical safety guidelines.

Version history

Version Date Author Change summary
v1.0 2025-11-20 Dillon Corvino Initial upgraded version for blood processing
v1.1 2025-11-26 Dillon Corvino Manual check and update protocol
v1.2 2026-09-18 Dillon Corvino Replaced # Buffers used (bare library(tidyverse), leaked R package-startup warnings into the rendered document) with an auto-generated # Buffers Required section, kept in its correct position before Materials; added BUF-FACS-BUF ID to the FACS Buffer reagent row.