EasySep™小鼠TIL（CD45）正选试剂盒
How to Isolate Mouse Bone Marrow Cells by Centrifugation
How to Isolate Mouse Bone Marrow Cells by Centrifugation
This protocol provides a simple and reliable method for isolating bone marrow cells from mouse femurs and tibias using centrifugation. The resulting single-cell suspension is compatible with downstream applications, including EasySep™ cell isolation which enables simple, column-free enrichment of target cell populations. Depending on the target cell type and downstream application (e.g. magnetic cell isolation, flow cytometry, cell culture, etc.), modifications such as red blood cell lysis or buffer selection may be required. Refer to the EasySep™ kit-specific Product Information Sheet (PIS) for recommended sample preparation guidelines.
Materials
- Mouse femurs and tibias
- Sterile razor blade or scalpel
- 18-gauge blunt-end needle
- 1.5 mL microcentrifuge tubes
- 0.5 mL microcentrifuge tubes
- 50 mL conical tubes (e.g. Falcon® Conical Tubes, Catalog #38010)
- 70 µm nylon mesh cell strainer (e.g. 70 µm Reversible Strainer, Large, Catalog #27260)
- Centrifuge (note: a refrigerated centrifuge is recommended when using pre-cooled buffer)
- Recommended medium according to your cell isolation kit's Product Information Sheet (PIS). Recommended options include:
- EasySep™ Buffer (Catalog #20144)
- RoboSep™ Buffer (Catalog #20104)
- Phosphate-buffered saline containing 2% fetal bovine serum and 1 mM EDTA
Protocol
- Harvest the femurs and tibias from the mouse.
- Carefully remove all surrounding muscle and tendon tissue using a sterile scalpel. Alternatively, gently rub the bones with a sterile alcohol wipe or paper towel to remove residual soft tissue.
- Cut the bones open at one end, i.e. remove the epiphysis, to expose the bone marrow.
- Punch a hole through the bottom of a 0.5 mL microcentrifuge tube using an 18-gauge blunt-end needle.
- Place the bones with the cut end facing down into the 0.5 mL tubes. Important: maximum two tibias and two femurs per tube.
- Add 100 µL of cold recommended medium to a 1.5 mL microcentrifuge tube.
- Place the 0.5 mL tube containing the bones (step 5) into the 1.5 mL tube.
- Centrifuge the bones for 1 minute at 2500 x g at room temperature or 4°C with the brake on.
- Inspect the bones for any remaining pink coloration. If present, repeat step 8 and re-cut the bone ends prior to the second centrifugation to improve marrow recovery.
- After centrifugation, the bone marrow will be at the bottom of the 1.5 mL tube. Discard the 0.5 mL tube and bones.
- Add 0.4 mL of recommended medium to the bone marrow and gently resuspend the cells by pipetting up and down several times to disperse any aggregates.
- Place a 70 µm cell strainer over a new conical tube.
- Pre-wet the 70 µm cell strainer using the recommended medium and filter the cell suspension to remove aggregates and debris. If applicable, pool cells from multiple tubes. Rinse the strainer with additional medium to maximize cell recovery.
- Top up the tube with medium and centrifuge at 300 x g for 10 minutes with the brake on to pellet the cells.
- Carefully remove the supernatant.
- Gently flick the tube to loosen the cell pellet.
- The cells are now ready for downstream applications. For cell separation, determine the total nucleated cell (TNC) number, then refer to the EasySep™ kit-specific Product Information Sheet (PIS) for the recommended cell concentration and resuspend the cells in the appropriate volume of the recommended buffer.
Did you know STEMCELL Technologies offers a broad portfolio of EasySep™ Mouse Cell Isolation Kits? Discover simplified column-free magnetic cell isolation of a wide range of mouse cell populations.
If you have any questions, contact us at techsupport@stemcell.com.
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