Me20.4 1 h4

Blood samples were collected from ten patients who underwent R0 resections on the day before surgery. As for the 13 patients who received CT or CRT, blood samples were obtained before the start of the therapy. Blood samples were collected from each donor in ethylene-diamine-tetra-acetic acid (EDTA)-2K Vacutainer Tubes (TERUMO, Tokyo, Japan). Specimens were maintained at 4 °C and processed within 4 h of phlebotomy. Mononuclear cells were enriched from 3 mL of peripheral blood using LymphoPrep™ (Nycomed AS, Oslo, Norway). These were incubated in the presence of allophycocyanin (APC)-conjugated monoclonal mouse anti-human EpCAM [clone HEA (human epithelial antigen)-125, dilution 1:100], fluorescein isothiocyanate (FITC)-conjugated monoclonal mouse anti-human p75 neurotrophic receptor (p75NTR, clone ME20.4-1.H4, dilution 1:50), FITC-conjugated monoclonal mouse anti-human CD44 (clone BD105, dilution 1:100, Miltenyi Biotec), or isotype-matched APC/FITC-conjugated antibodies (clone IS5-21F5, dilution 1:50) (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4 °C for 30 min. Samples were analyzed on the fluorescence-activated cell sorting (FACS)Canto II flow cytometer and sorted on a FACS Aria II flow cytometer using BD FACS Diva software (BD Biosciences, San Jose, CA, USA). Cells were stained with 7-aminoactinomycin D (7-AAD; Bio-Rad Laboratories, CA, USA) to exclude any dead cells.

To enrich LMP1-expressing cells, Jurkat cells co-transfected with pMACS-LNGFR were washed with PBS (without Ca²⁺ and Mg²⁺) 48 h post transfection, and stained with anti-LNGFR-PE conjugated antibodies (ME20.4-1.H4; 1:10; Miltenyi Biotec) for 10 min (4°C), followed by an incubation with anti-PE MicroBeads (Miltenyi Biotec) for 15 min (4°C). Labeled cells were separated using MACS LS columns (Miltenyi Biotec) on a MidiMACS™ Separator (Miltenyi Biotec). The percentage of cells stained for LNGFR was determined with the BD Accuri C6 flow cytometer (BD Biosciences) before and after magnetic separation.