Live dead fixable near infrared dye

Mouse peripheral blood was collected in tubes containing EDTA (ref: 41.1504.105, Sarstedt Inc.). Red blood cells were lysed using ACK lysis buffer once to twice. Remaining cells were resuspended in FACS buffer. Small aliquots were taken for cell counting. The rest of the samples were stained with the indicated markers for flow cytometry analysis. The live/dead fixable near‐infrared dye (ref: 65‐0865‐14, ThermoFisher Scientific) was used to exclude dead cells.

Livers were cut into small pieces of 2–4 mm and transferred to 6‐well plates containing 5 ml of 2 mg/ml collagenase D (ref: 11088866001, Roche) solution. Liver tissues were incubated at 37°C for 40 min and filtered through a 70‐μm cell strainer. After centrifugation (1,500 rpm, 5 min, 4°C), red blood cells were lysed using ACK lysis buffer. Samples were spun down at 1,500 rpm for 5 min, and single‐cell suspensions were prepared in FACS buffer and stained against the indicated markers for flow cytometric analysis. The live/dead fixable near‐infrared dye (ref: 65‐0865‐14, ThermoFisher Scientific) was used to exclude dead cells.

Tumors were cut into small pieces of 2–4 mm and transferred to gentleMACS C Tubes (ref: 130096334, Miltenyi Biotec) in 5 ml of CDTI buffer (RPMI supplemented with 2% heat‐inactivated FBS, 2 mg/ml collagenase D [ref: 11088882001, Roche], 40 U/ml DNase bovine pancreas grade I [ref: D4527‐40 KU, SIGMA‐Aldrich], and 1 mg/ml trypsin inhibitor [ref: T9003‐1G, Sigma‐Aldrich]). C‐tubes were placed onto gentleMACS Octo Dissociator, and GentleMACS program m_impTumor_03 was used, followed by 45‐min incubation at 37°C and GentleMACS program m_impTumor_01. After centrifugation (1,400 rpm, 7 min, 4°C), red blood cells were lysed using ACK lysis buffer. Samples were filtered through a 100‐μm cell strainer and spun down at 1,500 rpm for 7 min. Single‐cell suspensions were prepared in FACS buffer and stained against the indicated markers for flow cytometric analysis. The live/dead fixable near‐infrared dye (ref: 65‐0865‐14, ThermoFisher Scientific) was used to exclude dead cells.