Enrichment of bead- and label-free human tumor-infiltrating leukocytes (TILs) from human tumor tissue
Protocol
Preparation of buffer for cell enrichment and flow cytometry
Separation 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 autoMACS® 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.
Preparation of buffer for bead removal for REAlease® MicroBead Kits
REAlease Bead Release buffer: Prepare a 1:50 dilution of REAlease Bead Release Reagent (50×), e.g., for 1 mL add 20 μL of REAlease Bead Release Reagent to 980 μL of separation buffer.
▲Note: Use buffer freshly prepared at the same day. Store at room temperature.
▲Note: Prepare 1 mL per MS Column and 5 mL per LS Column.
Carefully create a single-cell suspension from a solid human tumor using the gentleMACS™ Octo Dissociator with Heaters in combination with the Tumor Dissociation Kit, human. For detailed instructions, follow the protocol of the kit data sheet.
- It is recommended to filter the magnetically labeled cell suspension to guarantee it is single-celled before separating it on the column.
- Enrich TILs using REAlease® TIL MicroBeads, human, and LS or MS Columns.
- For detailed instructions, refer to the protocol of the MicroBead kit data sheet.
- Place a Pre-Separation Filter (30 µm) on the LS or MS Column.
- Rinse the column 3× with PEB buffer and ensure that the filter is pre-wetted.
- Apply the cell suspension and washing buffer onto the filter on the column.
- Proceed with bead removal.
- (Optional) Perform removal of REAlease Complex for a second magnetic labeling.
View details
Materials
For tumor tissue dissociation
- Tumor Dissociation Kit, human (# 130-095-929)
- gentleMACS™ Octo Dissociator with Heaters (# 130-096-427)
- gentleMACS C Tubes (# 130-093-237)
- 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)
For enrichment and flow cytometry analysis of TILs
- Select your marker of interest among the following:
REAlease® CD45 (TIL) MicroBead Kit, human (# 130-121-563)
REAlease CD3 (TIL) MicroBead Kit, human (# 130-121-562)
REAlease CD4 (TIL) MicroBead Kit, human (# 130-121-559)
REAlease CD8 (TIL) MicroBead Kit, human (# 130-121-560)
REAlease CD4/CD8 (TIL) MicroBead Kit, human (# 130-121-561) - QuadroMACS™ Starting Kit (LS) (# 130-091-051)
- Separation 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 autoMACS® 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. - REAlease Bead Release buffer: Prepare a 1:50 dilution of REAlease Bead Release Reagent (50×), e.g., for 1 mL add 20 μL of REAlease Bead Release Reagent to 980 μL of separation buffer.
▲Note: Use buffer freshly prepared at the same day. Store at room temperature.
▲Note: Prepare 1 mL per MS Column and 5 mL per LS Column. - (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 recombinant antibodies for flow cytometry analysis, e.g., CD45 Antibody, anti-human, VioBlue®. Learn more about our antibodies for flow cytometry.
▲Note: Due to expression of Fcγ receptors on tumor-infiltrating leukocytes, REAfinity™ 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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