Immunophenotyping of hematopoietic stem cells (HSCs) from mouse bone marrow using flow cytometry
MBTP 21: Miltenyi Biotec-tested panel 21
MBTP 21: Miltenyi Biotec-tested panel 21
This application protocol describes the flow cytometric hematopoietic stem cell identification and analysis after isolation and enrichment of lineage-negative bone marrow cells from healthy C57BL/6 mice using the Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin (# 130-092-613).
Protocol
Surface staining
- Volumes given below were for up to 106 nucleated cells. When working with fewer than 106 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
- Cell number was determined.
- Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL buffer.
- 10 µL Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin was added to the cells.
- Cells were resuspended and incubated for 10 minutes in the dark at 4 °C.
- Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 10 minutes at 4 °C. Supernatant was aspirated completely.
- Two separate antibody staining cocktails were prepared according to the dilutions mentioned in the respective antibody data sheets: Staining cocktail 1 containing antibodies from panel 1 and staining cocktail 2 containing antibodies from panel 2.
- 100 µL staining cocktail 1 or 2 was added to the cells.
- Cells were resuspended and incubated for 10 minutes in the dark at 4 °C.
- Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL of PEB buffer and 1 µL of Propidium Iodide Solution was added right before flow cytometric analysis using the MACSQuant® Analyzer.
Antibody panels for hematopoietic stem cells
Antibody panel 1
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin | - | Cell type exclusion | - | - |
| Biotin | REA746 | - | VioBlue® | 450/50 (violet) |
| CD34 | REA383 | Cell type exclusion | FITC | 525/50 (blue) |
| CD135 (FLT3) | REA779 | Cell type exclusion | APC | 655-730 (red) |
| Sca-1 | REA422 | LSK cells | PE-Vio® 770 | 750LP (blue) |
| CD117 | 3C11* | LSK cells | PE | 585/40 (blue) |
| Propidium Iodide Solution | - | Viability | - | 655-730 (blue) |
*Alternative REAfinity™ Antibodies available: CD117 Antibody, anti-mouse, PE, REAfinity and CD48 Antibody, anti-mouse, Vio Bright B515, REAfinity.
Antibody panel 2
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin | - | Cell type exclusion | - | - |
| Biotin | REA746 | - | VioBlue | 450/50 (violet) |
| CD48 | HM48-1* | Cell type exclusion | FITC | 525/50 (blue) |
| CD150 (SLAM) | REA299 | Long-term HSC | APC | 655-730 (red) |
| Sca-1 | REA422 | LSK cells | PE-Vio 770 | 750LP (blue) |
| CD117 | 3C11* | LSK cells | PE | 585/40 (blue) |
| Propidium Iodide Solution | - | Viability | - | 655-730 (blue) |
*Alternative REAfinity Antibodies available: CD117 Antibody, anti-mouse, PE, REAfinity and CD48 Antibody, anti-mouse, Vio Bright B515, REAfinity.
Gating strategy
Materials
Antibody panels for hematopoietic stem cells
Antibody panel 1
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin | - | Cell type exclusion | - | - |
| Biotin | REA746 | - | VioBlue® | 450/50 (violet) |
| CD34 | REA383 | Cell type exclusion | FITC | 525/50 (blue) |
| CD135 (FLT3) | REA779 | Cell type exclusion | APC | 655-730 (red) |
| Sca-1 | REA422 | LSK cells | PE-Vio® 770 | 750LP (blue) |
| CD117 | 3C11* | LSK cells | PE | 585/40 (blue) |
| Propidium Iodide Solution | - | Viability | - | 655-730 (blue) |
*Alternative REAfinity™ Antibodies available: CD117 Antibody, anti-mouse, PE, REAfinity and CD48 Antibody, anti-mouse, Vio Bright B515, REAfinity.
Antibody panel 2
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin | - | Cell type exclusion | - | - |
| Biotin | REA746 | - | VioBlue | 450/50 (violet) |
| CD48 | HM48-1* | Cell type exclusion | FITC | 525/50 (blue) |
| CD150 (SLAM) | REA299 | Long-term HSC | APC | 655-730 (red) |
| Sca-1 | REA422 | LSK cells | PE-Vio 770 | 750LP (blue) |
| CD117 | 3C11* | LSK cells | PE | 585/40 (blue) |
| Propidium Iodide Solution | - | Viability | - | 655-730 (blue) |
*Alternative REAfinity™ Antibodies available: CD117 Antibody, anti-mouse, PE, REAfinity and CD48 Antibody, anti-mouse, Vio Bright B515, REAfinity.
Additional reagents used
- Phosphate-buffered saline (PBS), pH 7.2
- PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.
- Deionized or distilled water
- Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
- (Optional) MACSQuant Calibration Beads (# 130-093-607)
- (Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)
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