Enrichment of bead- and label-free human tumor-infiltrating leukocytes (TILs) from human tumor tissue


The materials and methods described in this protocol are for manual separation of TILs. 

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.


The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.
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