This application protocol describes the pre-enrichment of lineage marker-negative, bone marrow-derived cells and their phenotypic analysis using flow cytometry.
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").
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 data sheet.
▲ Note: Lineage depleted cells can be further enriched, e.g., to obtain LSK cells, using fluorescence-activated cell sorting.
▲ Note: It is recommended to filter 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 data sheet
Figure 1: Isolation of untouched lineage-negative cells from a mouse bone marrow cell suspension. After isolation with the DIrect Lineage Cell Depletion Kit, mouse, lineage-negative cells were fluorescently stained with the Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin and Biotin Antibody-APC and analyzed by flow cytometry using the MACSQuant® Analyzer. To evaluate the LSK (Lin–Sca-1+c-kit+) fraction, cells were further stained with CD117-PE (c-kit) and Sca-1 Antibody-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
Lineage-negative cells can be expanded in appropriate mouse HSC expansion medium supplemented with cytokines such as SCF, TPO, Flt3-Ligand, IL-3, and IL-6 according to standard cell culture protocols.
Lineage-negative cells can be further analyzed or sorted for HSC-specific surface markers by flow cytometry using MACS® Antibodies or recombinantly engineered REAfinity™ antibodies.
If cell separation was performed with the Direct Lineage Cell Depletion Kit, mouse, use Labeling Check Reagent, APC for the flow cytometric analysis. For separations performed with the Lineage Cell Depletion Kit, mouse, use Streptavidin, APC.
Prepare the staining cocktail as described in the following table:
|Reagent||Original cell fraction||Lineage-positive |
|Lineage-negative (depleted) cell fraction|
|Sca-1 Antibody-FITC||10 µL||10 µL||10 µL|
|CD117-PE||10 µL||10 µL||10 µL|
|Labeling Check Reagent, APC or Streptavidin, APC||10 µL||10 µL||10 µL|
|FcR Blocking Reagent||20 µL||20 µL||20 µL|
|PBE buffer||50 µL||50 µL||50 µL|
Use the MACS Flow Cytometry – Multicolor Panel Builder to quickly assemble the antibodies for analysis.
The following is a list of relevant resources that might be of interest:
|Column||Max. number of labeled cells||Max. number of total cells||Separator|
|Positive or negative selection|
|LS or Multi-24 Column Block (per column)||1×108||1×109||MultiMACS Cell24 Separator Plus|
|Positive or negative selection|
|autoMACS||2×108||4×109||autoMACS Pro, autoMACS|
▲ Note: Column adapters are required to insert certain columns into SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.
▲ Note: When using the Direct Lineage Cell Depletion Kit, the unwanted cell fraction is labeled and the target
cells remain unlabeled. Depending on the target cell frequency, the labeled fraction can represent the majority of the total cells. To avoid blocking of the column, do not exceed the maximum number of labeled cells per column. Estimate the number of labeled cells in the sample, split the sample if necessary and use the appropriate number of separation columns.
▲ Note: If separating with LS Columns and the MultiMACS Cell24 Separator Plus, use the Single-Column Adapter. Refer to the user manual for details.
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