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.
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).
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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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.
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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:
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.
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