Cellbanker2

Cells were cultured on 60-mm culture dishes with 4 mL of culture medium at 37 °C in 5%
CO₂. The culture medium consists of DMEM supplemented with 10% FBS, 100 U/mL
of penicillin, and 100 U/mL of streptomycin. The cells were passaged at least four times
and then frozen in a cryopreservation solution. As cryopreservation solutions, the culture
medium and 10% DMSO, Cell Banker 1, and Cell Banker 2 (Nippon Zenyaku Kogyo Co., Ltd.,
Fukushima, Japan) were used. One milliliter of a cell suspension containing 1 ×
10⁶ cells was quickly transferred to a 2.0-mL cryotube and frozen at a
cooling rate of 1 °C/min. After cooling to −80 °C, the cells were stored at −80 °C or in
LN₂ phase until use (typically over 8 years; HepG2 cells, August 16, 2008;
STO cells, September 12, 2008; HH cells, August 8, 2008; and NIH 3T3 cells, August 5,
2006).
The frozen tubes were placed in a water bath at 37 °C to thaw until the ice crystals had
nearly melted. The cell suspension was then diluted to 1:9 with ice-cold culture medium
and centrifuged at 1,000 rpm for 5 min. The supernatant was removed, and the cells were
resuspended in fresh medium. The cell viability was assessed using the trypan blue
exclusion test. The final concentration of trypan blue (GIBCO BRL; Life Technologies Inc.)
was 0.2%.

Peripheral blood mononuclear cell (PBMC) samples were collected prior to the first administration of the anti-PD-1 mAb using heparinized CPT Vacutainer tubes (Becton Dickinson Vacutainer Systems) as described previously (15 (link)). The samples were frozen using Cellbanker2 (Nippon Zenyaku Kogyo Co.) in a liquid nitrogen tank. For analyses of T-cell subsets, cells were incubated for 32–48 hours in a culture medium consisting of RPMI1640 and 10% FCS. Details of mass cytometry and flow cytometry analysis are shown in Supplementary Materials and Methods (Supplementary Table S1; Supplementary Fig. S1).