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The protocol below is a general procedure for the isolation, preparation, and staining of mouse bone marrow cells for flow cytometric analysis.

Materials

• Mouse femora and tibiae
• Phosphate-buffered saline (without Mg²⁺ and Ca²⁺; e.g. Catalog #37350) supplemented with 5 mM EDTA plus 1% fetal calf serum
• 21 - 26G needle
• 1 - 10 mL syringe
• Ammonium chloride solution (e.g. Catalog #07800)
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• 70 μm filter
• Anti-mouse CD16/CD32 antibody, Clone 2.4G2 (e.g. Catalog #60161)
• 7-AAD (e.g. Catalog #75001)
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Protocol

1. Isolate cells from mouse femora and tibiae by flushing bones with 1 - 3 mL phosphate-buffered saline (PBS) (without Mg2⁺ and Ca2⁺) supplemented with 5 mM EDTA plus 1% fetal calf serum. Flushing may be done using a 21 - 26G needle attached to a 1 - 10 mL syringe.
2. Generate a single-cell suspension by gently triturating the cells through the needle until large clumps are no longer present.
3. Lyse red blood cells (RBCs) by adding cold ammonium chloride solution and incubating bone marrow cells on ice for 10 minutes.
4. Following RBC lysis, top up the sample with an appropriate volume of PBS or similar wash buffer, and centrifuge at 300 x g for 5 minutes at room temperature (RT; 15 - 25°C). Resuspend the cell pellet in PBS and filter through a 70 μm filter to remove aggregated cell clumps and debris.
5. Count the cells in the filtered suspension and dilute to 1 x 10⁷ cells/mL in the same medium. The expected recovery of nucleated cells from both tibiae and femora (i.e. 4 long bones) of a normal male C57Bl/6 mouse is approximately 4 - 5 x 10⁷ cells.
6. To reduce non-specific binding of fluorescent antibodies, block F_c receptors by incubating cells with anti-CD16/32 (clone 2.4G2) antibody and 10% rat serum for 10 minutes on ice.
7. For single antibody-stained controls, aliquot 3 x 104 cells per tube and stain with optimized concentrations of individual antibodies in a total volume of ~100 μL.
8. In parallel, set up FMOs and test samples by aliquoting 2 - 3 x 10⁶ cells per tube and stain with optimized concentrations of antibodies.
9. Incubate cells for 20 minutes on ice, in the dark.
10. Wash cells in an appropriate volume of PBS and centrifuge at 300 x g for 5 minutes at RT, then aspirate the supernatant.
11. Resuspend cells in 100 μL PBS.
12. Just prior to acquisition, add 1 μg/mL 7-AAD to all samples, except unstained, single antibody-stained, and 7-AAD-FMO controls. Addition of 7-AAD to separate unstained and single antibody-stained controls should be considered if there are many dead or apoptotic cells that can cause increased autofluorescence or non-specific staining.
    
    
    Note: This general protocol uses 7-AAD to detect non-viable cells, but other viability dyes such as propidium iodide (PI) or amine-reactive dyes may also be used.
13. If using compensation beads, stain controls according to the manufacturer’s directions. To provide an optimal fluorescence signal, it is important to titrate the antibody so that the highest staining index is achieved.