Mouse Bone Marrow Isolation to Single-Cell Suspension

Dillon Corvino

Isolation of mouse bone marrow cells from long bones for flow cytometry, sorting, and downstream immunological assays.
Published

September 18, 2026

Protocol ID: MUS-BM-ISO
Version: v1.2
Author: Dillon Corvino

Purpose

This protocol describes the isolation of bone marrow cells from mouse long bones (typically femurs and tibias) by flushing with buffer, mechanical dispersion, and optional RBC lysis. The resulting suspension is suitable for flow cytometry, FACS sorting, and functional assays.

Critical notes (read before starting)

  • Work on ice or at 4 °C after marrow isolation to preserve viability.
  • Keep bones moist at all times in cold buffer; do not allow them to dry.
  • Use fresh, sterile instruments and avoid introducing bone shards into the final suspension.
  • If erythrocyte content is high, perform controlled RBC lysis; avoid prolonged exposure to lysis buffer.

Approximate timing

  • Bone dissection: 10–15 min per mouse (faster when batching)
  • Marrow flushing and dispersion: 10–15 min
  • RBC lysis and washing: 10–15 min

Total hands-on time per mouse: 30–45 minutes, depending on experience and batching.

Procedure

STEP 1 – Bone collection

  1. Euthanise the mouse using an approved method.
  2. Spray the hind limbs with 70% ethanol and transfer the carcass to a sterile working area (e.g. Class II biosafety cabinet, if available).
  3. Dissect out femurs using scissors and forceps.
  4. Remove all excess muscle and connective tissue from the bones.
  5. Place cleaned bones in a 50 mL tube or petri dish containing cold FACS Buffer or PBS and keep on ice.
Tip

Note how many bones per mice for later cell number calculations

STEP 2 – Bone marrow extraction

Method 1 - Flushing the bone marrow

  1. In a petri dish or on a sterile surface, cut off the epiphyses (ends) of each bone with sterile scissors to expose the marrow cavity.
  2. Fill a 10 mL syringe fitted with an appropriate gauge needle (e.g. 25 G) with cold FACS Buffer or PBS.
  3. Position the needle into one end of the bone and flush the marrow into a petri dish or 50 mL tube:
    • Use ~1–3 mL buffer per bone, depending on size.
  4. Repeat flushing from both ends if necessary until the bone appears pale and empty.
  5. Gently triturate the flushed marrow with a 5–10 mL pipette or by drawing up and down with the syringe (without needle) to break up clumps.
Warning

Avoid vigorous pipetting that shears cells; use gentle, repeated strokes to obtain a single-cell suspension.

Method 2 - Centrifugation

  1. Poke a small hole (using a needle) in a PCR tube
  2. Place PCR tube in an Eppendorf tube
  3. Add 200 µL cold FACS Buffer
  4. Cut off the epiphyses (ends) of each bone with sterile scissors to expose the marrow cavity.
  5. Spin in table-top micro-centrifuge for 30 sec - 2 mins
  6. Ensure bone marrow has not gotten stuck in the PCR tube and bones appear pale and empty (Repeat spin as needed)

STEP 3 – Filtration and initial wash

  1. Pass sample through a 70 µm cell strainer onto a 15 mL Falcon tube.
  2. Rinse the petri dish/source tube and strainer with additional cold FACS Buffer to collect remaining cells and top up to 15 mL.
  3. Centrifuge at 400 g for 5 minutes at 4 °C.
  4. Carefully discard the supernatant.

STEP 4 – RBC lysis (if required)

  1. Gently resuspend the bone marrow pellet in 100 µL 1× RBC Lysis Buffer (e.g. HybriMax or equivalent).
  2. Incubate for 5 minutes at room temperature, gently inverting the tube once or twice.
Warning

Do not exceed 5 minutes of RBC lysis; prolonged exposure can reduce leukocyte viability and alter surface marker expression.

  1. After lysis, immediately top up the tube to 10 mL with FACS Buffer.
  2. Centrifuge at 400 g for 5 minutes at 4 °C.
  3. Carefully discard the supernatant.

STEP 5 – Final wash and resuspension

  1. Resuspend cells in 1 mL FACS Buffer (adjust volume according to desired final concentration and downstream application).
  2. Gently flick or briefly vortex to loosen the pellet.
  3. If residual clumping is observed, pass the suspension again through a 70 µm strainer into a fresh tube.
  4. Keep the suspension on ice until counting, staining, or further processing.

STEP 6 – Downstream applications

Proceed to:

  • Cell counting and viability assessment.
  • Flow cytometry staining panels (e.g. hematopoietic profiling).
  • FACS sorting (e.g. HSCs, progenitors, mature lineages).
  • Functional assays or in vitro culture as per experimental design.

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
FACS Buffer In-house (see buffer) BUF-FACS-BUF; cold; used for flushing and washes
PBS 1× (sterile) Any Optional; for flushing
RBC Lysis Buffer (1×) [TBD] 1–2 mL per pellet; as needed
70% Ethanol Any For surface disinfection of carcass

Disposables

Item Notes
50 mL Falcon tubes For bone storage and processing
15 mL Falcon tubes Optional; for smaller volumes
70 µm cell strainers For debris removal
10 mL syringes + needles (~25 G) For marrow flushing
5–10 mL serological pipettes For trituration
Petri dishes For bone trimming and flushing
Gloves, bench wipes Standard PPE and cleanup

Equipment

Equipment Notes
Biosafety cabinet (Class II) Recommended for tissue handling
Benchtop centrifuge 400 g at 4 °C, compatible with 50 mL tubes
Dissection scissors and forceps For bone isolation and cleaning
Timer For RBC lysis timing

Troubleshooting

Issue Possible cause Suggested solution
Low cell yield Incomplete marrow flushing Flush from both ends; increase volume per bone slightly
Excess debris or bone fragments Inadequate filtration Ensure proper use of 70 µm strainer; repeat filtration if needed
High red cell contamination Insufficient RBC lysis Repeat RBC lysis briefly (≤3 min) followed by wash
Poor viability Overly vigorous trituration or long RT handling Use gentler pipetting; keep steps cold; minimise processing time

Safety (brief)

  • Handle mouse tissues and sharps according to institutional S1/S2 and animal ethics guidelines.
  • RBC lysis buffers may contain hazardous components; avoid skin/eye contact and dispose of waste according to chemical safety rules.
  • Bone fragments and needles must be discarded into appropriate sharps containers.

Version history

Version Date Author Change summary
v1.0 2025-11-20 Dillon Corvino Initial upgraded version for bone marrow isolation
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.