PBE buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5 % bovine serum albumin (BSA), and 2  mM EDTA by diluting MACS® BSA Stock Solution 1:20 with autoMACS® Rinsing Solution. Keep buffer cold (2−8 °C). Degas buffer before use.

Isolate mononuclear cells from bone marrow extracted from mouse femoral bones.

▲Note: All steps should be performed on ice cold PBE buffer (see "Things to prepare in advance").

1. Extract mouse limbs and place on cold PBE buffer.
2. Use sterile scissors and forceps to isolate femoral bones. Place them in a 6 cm cell culture dish with 2–3 mL ice cold PBE buffer.
3. Isolate bone marrow cells either by grinding isolated femoral bones in a mortar or by flushing the cells out of the bone with a syringe.
4. Pass cells through a 30 µm MACS® SmartStrainer into a 50 mL centrifuge tube to remove cell clumps that can clog the column. Wet the filter with PBE buffer before adding the cells.
5. Adjust final volume of cell suspension to 15–30 mL, depending on estimated cell number.
6. Take 10 µL of cell suspension to determine cell number using a Neubauer chamber or another cell counting device.
7. Store a 200 µL aliquot of the cell suspension on ice for later flow cytometry analysis.
8. Centrifuge the 50 mL centrifuge tube containing the isolated bone marrow cells at 400×g for 10 minutes.

Deplete cells expressing lineage markers from total bone marrow mononuclear cells using either the Direct Lineage Cell Depletion Kit, mouse or the Lineage Cell Depletion Kit, mouse. The first generates a high cell yield. The latter enables a more stringent depletion. Follow the protocol of the corresponding kit data sheet.

▲ Note: Lineage depleted cells can be further enriched, e.g., to obtain LSK cells, using fluorescence-activated cell sorting.

▲ Note: We recommend filtering the magnetically labeled cells before separation to guarantee a single-cell suspension either manually or via the automated protocol using the autoMACS® Pro Cell Separator. 

Download kit data sheet

Lineage Cell Depletion Kit, mouse

Direct Lineage Cell Depletion Kit, mouse

Lineage marker-negative cells can be further analyzed or sorted for HSC-specific surface markers by flow cytometry using MACS® Antibodies or recombinant engineered REAfinity™ antibodies.

If cell separation was performed with the Direct Lineage Cell Depletion Kit, mouse, use Labeling Check Reagent-APC for the flow cytometry analysis. For separations performed with the Lineage Cell Depletion Kit, mouse, use Streptavidin-APC.

Prepare the staining cocktail as described in the following table:

1. Centrifuge aliquots of the different cell fractions (original, positive and negative fractions) at 300×g for 10 minutes.
2. Discard supernatant.
3. Resuspend each cell fraction in 100 µL of staining cocktail. Carefully pipet the suspension up and down.
4. Incubate the resuspended cells at 4 °C for 10 minutes.
5. Add 100 µL PBE buffer to each cell fraction and mix thoroughly by pipetting up and down.
6. Centrifuge cells at 300×g for 10 minutes.
7. Resuspend cell fractions in 200 µL PBE buffer for flow cytometry analysis.

The following is a list of relevant resources that might be of interest:

• REAfinity™ Recombinant Antibodies – Flow cytometry is in their genes
  

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