Application protocol

Pre-enrichment of mouse hematopoietic stem and progenitor cells

In this application protocol, we describe the pre-enrichment of lineage marker-negative, bone marrow-derived cells, and their phenotypic analysis using flow cytometry.

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Protocol

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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.

1. Things to prepare in advance

Preparation of buffer for cell isolation and depletion, and for 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.

2. Isolation of mononuclear cells

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").

Extract mouse limbs and place on cold PBE buffer.
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.
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.
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.
Adjust final volume of cell suspension to 15–30 mL, depending on estimated cell number.
Take 10 µL of cell suspension to determine cell number using a Neubauer chamber or another cell counting device.
Store a 200 µL aliquot of the cell suspension on ice for later flow cytometry analysis.
Centrifuge the 50 mL centrifuge tube containing the isolated bone marrow cells at 400×g for 10 minutes.

3. Depletion of lineage marker-positive cells

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

Before separation

After separation

LSK staining

Before separation

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After separation

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LSK staining

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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 Lineage Cell Detection Cocktail-Biotin and Anti-Biotin-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 Anti-Sca-1-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

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Clear separation of lineage-negative and lineage-positive cells. Untouched lineage negative cells from a mouse bone marrow cell suspension using the Lineage Cell Depletion Kit and a MidiMACS™ Separator with an LS Column. Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.

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Isolation of lineage^–/CD117⁺ cells from a mouse bone marrow cell suspension. After depletion of lineage⁺ cells using the Lineage Cell Depletion Kit, CD117 MicroBeads and a MidiMACS Separator with LS Columns were used to isolate CD117⁺ cells. The cells were then fluorescently stained with CD117-PE, and a panel of biotinylated antibodies against lineage markers and Anti-Biotin-APC. Cell debris and dead cells were excluded from the analysis.

4. Expansion of lineage marker-negative cells or LSK cells

Lineage marker-negative cells can be expanded in appropriate mouse HSC expansion media supplemented with cytokines such as SCF, TPO, FLT-3L and IL-3 and IL-6 according to standard cell culture protocols.

5. Flow cytometry analysis or further enrichment of lineage marker-negative cells

Staining cocktail preparation

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:

Reagent	Original cell fraction	Lineage marker-positive cell fraction	Lineage marker-negative (depleted) cell fraction
Anti-Sca-1-FITC	10 µL	10 µL	10 µL
CD117-PE10	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 your analysis.

Staining procedure

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

Resources

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

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

Reagents and instrumentation requirements

General reagent and instrument requirements

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 (# 130-091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Keep buffer cold (2−8 °C). Degas buffer before use, as air bubble could block the column.

For mononuclear cell isolation

MACS SmartStrainers (30 µm) (# 130-098-458)
Neubauer chamber or other cell counting device

For lineage cell depletion

Lineage Cell Depletion Kit, mouse (# 130-090-858) or Direct Lineage Cell Depletion Kit, mouse (# 130-110-470)
▲ Note: Use the Direct Lineage Cell Depletion Kit for a high cell yield, or the Linear Cell Depletion Kit for a more stringent depletion.
(Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., Anti-Biotin-APC (# 130-113-288), CD117-PE (# 130-122-937) or CD117-APC (# 130-122-948). For more information about antibodies refer to www.miltenyibiotec.com/antibodies.
(Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometric exclusion of dead cells.
(Optional) Dead Cell Removal Kit (# 130-090-101) for the depletion of dead cells.
(Optional) Pre-Separation Filters, 30 µm (# 130-041-407) to remove cell clumps.
MACS Columns and MACS Separators: Choose the appropriate MACS Separator and MACS Columns according to the number of labeled cells and the number of total cells. Use autoMACS for automated magnetic cell separation.
▲ Note: Use LS Columns for the Direct Lineage Cell Depletion Kit, mouse. The kit can be additionally used with the MultiMACS™ Cell24 Separator Plus, and includes a completely automated cell isolation protocol for use with the autoMACS Pro.

Column	Max. number of labeled cells	Max. number of total cells	Separator
Positive or negative selection
MS	1×10⁷	2×10⁸	MiniMACS™, OctoMACS™, VarioMACS, SuperMACS II
LS	1×10⁸	2×10⁹	MidiMACS™, QuadroMACS™, VarioMACS, SuperMACS II
LS or Multi-24 Column Block (per column)	1×10⁸	1×10⁹	MultiMACS Cell24 Separator Plus
XS	1×10⁹	2×10¹⁰	SuperMACS II
Positive or negative selection
autoMACS	2×10⁸	4×10⁹	autoMACS Pro, autoMACS

▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or 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.

For expansion of lineage marker-negative cells or LSK cells

Mouse SCF, research grade (# 130-094-079)
Mouse Flt3-Ligand, research grade (# 130-094-038)
Mouse Thrombopoietin (TPO), research grade (# 130-094-083)
Mouse IL-3 IS, research grade (# 130-096-687)
Mouse IL-6, research grade (# 130-094-065)

For flow cytometry analysis

Anti-Sca-1-FITC, mouse (clone: D7) (# 130-102-297)
CD117-PE, mouse (clone: REA791) (# 130-111-693)
Labeling Check Reagent-APC (# 130-098-892) or Streptavidin-APC (# 130-106-792)
FcR Blocking Reagent, mouse (# 130-092-575)
MACSQuant® Analyzer 10

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Application protocol

Pre-enrichment of mouse hematopoietic stem and progenitor cells

Protocol

Materials

Protocol

1. Things to prepare in advance

Preparation of buffer for cell isolation and depletion, and for flow cytometry

2. Isolation of mononuclear cells

3. Depletion of lineage marker-positive cells

Direct Lineage Cell Depletion Kit, mouse

4. Expansion of lineage marker-negative cells or LSK cells

5. Flow cytometry analysis or further enrichment of lineage marker-negative cells

Staining cocktail preparation

Staining procedure

Resources

Materials

Reagents and instrumentation requirements

General reagent and instrument requirements

For mononuclear cell isolation

For lineage cell depletion

For expansion of lineage marker-negative cells or LSK cells

For flow cytometry analysis

Trademarks and disclaimers

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