Application protocol

Viral transduction of mouse lineage-negative hematopoietic stem and progenitor cells

 In this application protocol, we describe 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. 

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.

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) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., Anti-Biotin-APC (# 130-090-856), CD117-PE (# 130-102-795) or CD117-APC (# 130-102-796). 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.
  • 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. 
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive or negative selection
MS1×1072×10⁸MiniMACS™, OctoMACS™,
VarioMACS, SuperMACS II
LS1×1082×109MidiMACS™, QuadroMACS™,
VarioMACS, SuperMACS II
LS or Multi-24 Column Block (per column)1×1081×109MultiMACS Cell24 Separator Plus
XS1×1092×1010SuperMACS II
Positive or negative selection
autoMACS2×1084×109autoMACS 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 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 cytometry analysis of isolated lineage-negative cells

  • CD45-VioBlue®, mouse  (# 130-110-664)
  • CD45RA-FITC, mouse  (# 130-109-728)
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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.

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 data sheet below.

Download data sheet

Isolation of mononuclear cells from bone marrow aspirates

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

Direct Lineage Cell Depletion Kit, mouse
Before separation
After separation
LSK staining

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.

Transduce the lineage marker-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, FLT-3L and 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

Vectofusion-1

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.

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.  

Use the MACS Flow Cytometry – Multicolor Panel Builder to quickly assemble the antibodies for your analysis.

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

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