Purified anti-human Ki-67 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Intracellular staining of Ki-67 was performed using a FoxP3 transcription factor staining buffer set (eBioscience) and specific antibodies.	All stained samples were acquired with a LSR II flow cytometer (BD Biosciences), and the data were analyzed by FlowJo software version 10.4.0 (Treestar).

Protocol tips
Intracellular staining of Ki-67 was performed using a FoxP3 transcription factor staining buffer set (eBioscience) and specific antibodies.
Downstream tips
All stained samples were acquired with a LSR II flow cytometer (BD Biosciences), and the data were analyzed by FlowJo software version 10.4.0 (Treestar).

Publication protocol

Multi-color flow cytometry of PBMCs was performed at Korea Advanced Institute of Science and Technology (Daejeon, Republic of Korea), with the investigators blinded to the clinical outcome of the patients. The following fluorochrome-conjugated mAbs were used in multi-color flow cytometry: anti-CD8 (SK1 and RPA-T8), anti-CD3 (HIT3a), anti-CD4 (SK3), and anti-CD28 (CD28·2) from BD Biosciences; anti-PD-1 (EH·12·2H7) and anti-Ki-67 (Ki-67) from BioLegend; anti-CD14 (61D3) and anti-CD19 (HIB19) from eBioscience; and anti-human IgG4 Fc (HP6025) from Southern Biotech. The therapeutic binding of pembrolizumab or nivolumab (human IgG4) to PD-1+ cells interfered with PD-1 staining of posttreatment specimens. Therefore, we performed antihuman IgG4 Fc staining in addition to anti-PD-1 staining to define PD-1+ cells. Dead cells were identified using the LIVE/DEAD Fixable Red Dead Cell Stain Kit (Invitrogen). Intracellular staining of Ki-67 was performed using a FoxP3 transcription factor staining buffer set (eBioscience) and specific antibodies. Tumor antigen–specific CD8+ T cells were detected by a PE-conjugated MHC-I dextramer (HLA-A*0201: NY-ESO-1157; Immudex). HCMV-specific CD8+ T cells were detected by PE-conjugated MHC-I pentamer (HLA-A*0201: pp65495; Proimmune). All stained samples were acquired with a LSR II flow cytometer (BD Biosciences), and the data were analyzed by FlowJo software version 10.4.0 (Treestar). The gating strategies are summarized in Supplementary Fig. S1.

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