Application protocol

Isolation, expansion, and analysis of human hematopoietic stem cells (HSC)

In this application protocol, we describe how human HSCs can be isolated form various sources, such as bone marrow or cord blood, expanded in highly defined cell culture conditions, and analyzed by flow cytometry or cell culture assays. 

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 2mM 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).
  • 15 mL of Ficoll-Paque™ (ρ = 1.077 g/mL)
  • MACS® SmartStrainers (100 µm) (# 130-098-463)

For CD34+ cell isolation

  • CD34 MicroBead Kit UltraPure, human (# 130-100-453)
  • MC CD34 Stem Cell Cocktail (# 130-093-427) for flow cytometry analysis of separated cells. 
  • (Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., CD34-FITC (# 130-081-001), CD34-PE (# 130-081-002), CD34-APC (# 130-090-954), CD133 (293C3)-PE (# 130-090-853), CD133 (293C3)-PE (# 130-090-854), CD45-FITC (# 130-080-202), CD45-PE (# 130-080-201), or CD45-APC (# 130-091-230). 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: CD34+ cells can be enriched using MS, LS, or XS Columns (positive selection). Cells that express the CD34 antigen strongly can also be depleted using MS, LS, or XS Columns.
    ▲ Note: Positive selection or depletion can be automated using the autoMACS Pro or the autoMACS Separator.
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
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.

For CD34+ cell expansion

  • StemMACS™ HSC Expansion Media XF, human (# 130-100-463)
  • StemMACS HSC Expansion Cocktail (# 130-100-843)

Cytokines included in the StemMACS HSC Expansion Cocktail:

  • Human SCF (# 130-096-693)
  • Human FLT3-Ligand (# 130-096-476)
  • Human Thrombopoietin (TPO) (# 130-094-011)

For flow cytometry analysis

  • StemMACS HSC-CFU complete with Epo, human (# 130-091-280)
  • CD45-VioBlue, human  (# 130-092-880)
  • CD45RA-FITC, human  (# 130-092-247)
  • CD133/2 (293C3)-PE, human (# 130-090-853)
  • CD34-APC, human (# 130-090-954)
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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.