Application protocol

Enrichment of tumor-infiltrating leukocytes (TILs) from mouse tumor tissue

Tumor-infiltrating leukocytes (TILs) represent a minority of the complete cell population within tumor tissue. In this application protocol, we describe a time-saving workflow that overcomes this limitation and increases sensitivity of downstream analyses. Mouse tumor tissue is dissociated into a viable single-cell suspension and TILs (CD45+) are enriched via MACS® Technology.

Protocol

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The following instructions are for manual cell enrichment.

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 tumor tissue dissociation

  • Tumor Dissociation Kit, mouse (# 130-096-730)
  • gentleMACS™ Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237)
  • RPMI 1640 (#130-091-440) or DMEM (# 130-091-437) culture media
  • MACS® SmartStrainers (70 µm) (# 130-098-462)
  • MACSmix™ Tube Rotator (# 130‑090‑753) in combination with an incubator at 37 °C
  • (Optional) Red Blood Cell Lysis Solution (10×) (# 130-094-183)
  • (Optional) MACS Tissue Storage Solution (# 130-100-008) 
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.

For enrichment and flow cytometry analysis of TILs

  • CD45 (TIL) MicroBeads, mouse (# 130-110-618)
  • QuadroMACS™ Starting Kit (LS) (# 130-091-051
  • PBE buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS BSA Stock Solution (# 130‑091‑376) 1:20 with automMACS® Rinsing Solution (#130-091-222). Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column. Always use freshly prepared buffer.
    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 mouse serum albumin, mouse serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
  • (Optional) Pre-Separation Filters (30 µm) (#130-041-407) to remove cell clumps
  • (Optional) MACS SmartStrainers (30 µm) (130-098-458) to remove cell clumps
  • (Optional) Fluorochrome-conjugated REA (REAfinity™ antibodies: recombinantly engineered, lacking Fcγ-binding site) CD45 antibodies for flow cytometry analysis, e.g., CD45-VioBlue®. Learn more about our antibodies and dyes.
    Note: Due to expression of Fcγ receptors on tumor-infiltrating leukocytes, REA antibodies are recommended.
  • (Optional) Propidium Iodide Solution (# 130-093-233), DAPI Staining Solution (# 130-111-570), 7-AAD Staining Solution (# 130-111-568), or Viobility™ Fixable Dyes (# 130-109-812, # 130-109-814, # 130-109-816) for flow cytometric exclusion of dead cells.
  • (Optional) Dead Cell Removal Kit (# 130-090-101) for the depletion of dead cells

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