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
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
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.
- Place a Pre-Separation Filter (30 µm) on the LS or MS Column.
- Rinse the column 3 times with PBE buffer, ensuring that the filter is pre-wetted.
- Apply the cell suspension and washing buffer to the filter on the column.
Download the data sheet
View details
The following is a list of additional resources that may be of interest:
- Preservation of cell surface epitopes
- Convenient tumor dissociation
- REAfinity™ Antibodies – Flow cytometry is in their genes
- REAfinity Antibodies – Frequently asked questions
- Brauner, J. et al. (2017) Workflow automation and parallelization improves the isolation and analysis of tumor-infiltrating immune cell populations. AACR, Washington D.C., USA.
- Miltenyi Biotec product list: Cancer antibodies, mouse
- Application Note: Functional TILs from human renal cell carcinoma. Baldan, V. et al., Manchester Academic Healthcare Science Centre.
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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Materials
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
