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.

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

Automated protocol

The materials and methods described in this Application are for the manual protocol. Automated cell isolation can be performed with the autoMACS® Pro or the multiMACS™ Cell24 instruments.
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Protocol


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The following instructions are for manual cell enrichment.

Preparation of buffer for cell enrichment and flow cytometry

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 1:20 with autoMACS Rinsing Solution. Keep buffer cold (2−8 °C). Prepare buffer fresh and degas prior to use.
▲ 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.

Create a viable single-cell suspension from a solid mouse tumor using the gentleMACS™ Octo Dissociator with Heaters in combination with the Tumor Dissociation Kit, mouse. Follow the protocol of the kit data sheet.

Download data sheet

Tumor Dissociation Kit, mouse

Enrich tumor-infiltrating leukocytes using CD45 (TIL) MicroBeads, mouse, and LS or MS Columns. Follow the protocol of the kit data sheet.

We recommend filtering the magnetically labeled cell suspension to guarantee it is single-celled before separating it on the column.

  1. Place a Pre-Separation Filter (30 µm) on the LS or MS Column.
  2. Rinse the column 3 times with PBE buffer, ensuring that the filter is pre-wetted.
  3. Apply the cell suspension and washing buffer to the filter on the column.

Download the data sheet

CD45 (TIL) MicroBeads, mouse

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Subpopulations of tumor-infiltrating leukocytes in B16F10 tumor tissue without prior enrichment.

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Subpopulations of tumor-infiltrating leukocytes in B16F10 tumor tissue after enrichment.

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