JNAR Protocol

Isolation of Mouse Bone Marrow Cells for BMDM Differentiation

Núria Crua· Postdoc· University of Lausanne (UNIL)

Published Jul 28, 2026

~0m total· 0 contributions

Cite this protocol in your article

JNAR-ID: JNAR-14608056

Copied to clipboard

PublishedLast updated Jul 28, 2026

Objective

To isolate viable bone marrow cells from mouse femurs and tibias using a centrifugation-based extraction method for downstream applications such as bone marrow-derived macrophage (BMDM) differentiation, flow cytometry, RNA extraction, and functional assays.

Classification

Sample typeCells

TechniqueCell Culture

Experimental contextIn vitroEx vivo

Biological modelMouse

Reproducibility status

Reproducible

No reproducibility blockers or risks reported. The protocol runs end-to-end.

15 healthy

⏱ Total —3 sections15 steps

Preparation of the Bone Marrow Collection Tubes

Using a sterile needle, create a small hole in the bottom of a 0.5 mL microcentrifuge tube.

Insert the pierced 0.5 mL tube into a sterile 2 mL microcentrifuge tube, which will serve as the collection tube during centrifugation.

Place one half femur and one half tibia into each pierced tube, ensuring that the cut ends of the bones are facing downward toward the hole.

Bone Marrow Isolation

Centrifuge the nested tubes at 13,000 × g for 15 seconds at room temperature.

Critical stepIncorrect centrifugation parameters may fail to pellet bone marrow cells or damage them.

Carefully remove and discard the pierced 0.5 mL tube containing the empty bones.

A compact bone marrow pellet should be visible at the bottom of the 2 mL collection tube.

Resuspend the pellet in 1.5 mL cold RPMI-1640.

Transfer the cell suspension through a 70 µm cell strainer into a 50 mL Falcon tube.

Wash the cell strainer with additional cold RPMI-1640 to obtain a final volume of 10 mL (approximately 8.5 mL wash volume).

Mix the suspension thoroughly by gentle pipetting.

Count the number of cells (usually diluting the sample 1:10).

Critical stepAccurate cell counting is essential for seeding the correct number of cells for culture.

Cell Counting and Seeding Table

Mice	Counting (cells/mL)	Total Cells number	Viability (%)	Volume for Culture (2.5 x 10⁶ cells)
1
2

BMDM Cell Culture

Transfer 2.5 × 10⁶ bone marrow cells into each T-25 tissue culture flask.

Critical stepSeeding the correct cell number is critical for consistent BMDM differentiation.

Add 7 mL of complete RPMI medium supplemented with:

13.1

Penicillin/Streptomycin (1×)

13.2

TGF-β (10 ng/mL final concentration) (7 µL of a 10 µg/mL stock; 1:1000 dilution)

13.3

GM-CSF (5 ng/mL final concentration) (3.5 µL of a 10 µg/mL stock; 1:2000 dilution)

Gently resuspend the cells by carefully rocking the flask.

Incubate at 37°C in a humidified incubator with 5% CO₂.