Application protocol
Viral transduction of mouse lineage-negative hematopoietic stem and progenitor cells
This application protocol describes the depletion of murine lineage cells from bone marrow samples, viral transduction of the remaining bone marrow cells, and their phenotypic analysis using flow cytometry.
Protocol
Preparation of buffers for cell isolation and depletion and for flow cytometry
PE buffer for isolation of mononuclear cells
Prepare a solution with phosphate buffered saline (PBS), pH 7.2, and 2 mM EDTA. Keep buffer cold (2–8 °C).
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
PBE buffer for cell depletion and flow cytometry
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) and degas before using.
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as respective serum albumin, respective serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
Isolate mononuclear cells by density gradient centrifugation using Ficoll-Paque™. Follow the Ficoll-Paque™ protocol from the special protocol below.
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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.
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Direct Lineage Cell Depletion Kit, mouse
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Before separation
After separation
LSK staining
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 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.
Transduce lineage-negative cells with a retroviral or lentiviral vector, either directly after MACS® Enrichment or after pre-stimulation culture in appropriate cell culture medium supplemented with cytokines such as SCF, TPO, Flt3-Ligand, IL-3, and IL-6.
Use Vectofusion-1®, a novel transduction enhancer, to boost transduction efficiency. Follow the protocol of the data sheet.
Download data sheet
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.
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:
- StemMACS™ HSC Expansion Media XF – A xeno-free culture system for hematopoietic stem cells
- CD34 MicroBead Kit UltraPure, human – Leading the way in magnetic HSC separation
- REAfinity™ Recombinant Antibodies – Flow cytometry is in their genes
- REAfinity Recombinant Antibodies – Frequently Asked Questions
- Nölle, V. et al., (2016) Recombinant antibodies for improved standardization in flow cytometry.
- Watts, M.J. et al., (2008) Performance of commercial methylcellulose media for short-term colony assays in routine transplantation practice. ISCT 2008. Miami, Florida.
- StemMACS™ HSC-CFU Media – Need to estimate HSC differentiation potential? We have the media!
Materials
For mononuclear cell isolation
- PE buffer: Phosphate buffered saline (PBS), pH 7.2, and 2 mM EDTA. Keep buffer cold (2–8 °C).
- 15 mL of Ficoll-Paque™ (ρ = 1.077 g/mL)
- MACS® SmartStrainers (100 µm) (# 130-098-463)
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) Hematopoietic Lineage Labeling Cocktail, anti-mouse, Biotin (# 130-092-613)
- (Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., Biotin Antibody, anti-mouse, APC, CD117 Antibody, anti-mouse, PE, or CD117 Abtibody, anti-mouse, APC. For more information about antibodies refer to www.miltenyibiotec.com/antibodies.
- (Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD Staining Solution (# 130-111-568) 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.
- 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.
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use. - 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™, SuperMACS II |
| LS | 1×108 | 2×109 | MidiMACS™, QuadroMACS™, 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 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 viral transduction and cell in vitro expansion
- 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)
- Vectofusin-1® (# 130-111-163)
For flow cytometric analysis of isolated lineage-negative cells
- CD45 Antibody, anti-mouse, VioBlue®, REAfinity™
- CD45RA Antibody, anti-mouse, FITC, REAfinity
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