Peripheral blood was collected at baseline (pre-treatment), C2D1, and post-cycle 3 (C4D1 or C6D1) for flow cytometry analysis. Peripheral blood was obtained using heparin collection tubes, and peripheral blood mononuclear cells (PBMCs) were isolated within 6 h using Ficoll-Paque Separation according to manufacturer’s instructions (GE Healthcare). PBMCs were cryopreserved in 10% DMSO, 90% FBS and thawed rapidly for flow cytometry analysis. Single-cell suspensions were prepared on ice in 2% FBS in PBS. Antibody cocktails were diluted in Brilliant Violet Buffer (BD Biosciences). Samples were acquired using a Cytek Aurora spectral cytometer with user settings established by Spectroflo QC beads. Unmixing and compensation were performed in Spectroflo software using a mix of reference controls from either single stained PBMCs or single stained OneComp compensation beads (eBioscience). Samples were stained with fluorescently tagged antibodies (Supplemental Table 4). Antibodies were titrated for optimal signal-to-noise ratio prior to use. Flow cytometry analysis was performed using Flowjo vX, and the CATALYST R package was used for FlowSOM analysis and UMAP projections [21 (link)]. All samples were gated on live, single cells.
Onecomp compensation beads
Manufactured by Thermo Fisher Scientific
OneComp compensation beads are a laboratory tool used to set up compensation on flow cytometers. They provide a standardized method for adjusting the electronic signal from different fluorophores to account for spectral overlap, ensuring accurate and reliable data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Brilliant violet buffer, by BD (3 mentions)
Cs t beads, by BD (3 mentions)
Fortessa, by BD (2 mentions)
Ficoll paque separation, by GE Healthcare (1 mentions)
Facsdiva v6, by BD (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Golgiplug, by BioLegend (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
4 protocols using onecomp compensation beads
1
Multiparametric Flow Cytometry Analysis
2
Quantification of Exhausted CD8+ T Cells
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PD-1+ CD39+ CD8+ Tex were quantified by flow cytometry as described before.30 Briefly, single-cell suspensions were stained at room temperature in PBS with 2% FBS. Optimized antibody cocktails contained antibodies to identify Tex within tumour infiltrating T cells: CD3 (UCHT1, RRID: AB_893299), CD8 (SK1 RRID: AB_10551438), CD45RA (HI100 RRID: AB_647424), CCR7 (G043H7 RRID: AB_2561753), PD-1 (EH1.2 RRID: AB_2033989), CD39 (A1 RRID: AB_940423). All antibodies were titrated for optimal signal:noise ratios using peripheral blood mononuclear cells. The acquisition of samples was performed on a BD Biosciences Fortessa instrument operated through FACSDiva 6.1.3. Photomultiplier tube voltages were established using BD Biosciences CS&T Beads. Compensation was calculated with single-stained OneComp compensation beads (eBioscience, Thermo Fisher Scientific). Tex abundance was quantified based on the proportion of PD1+CD39+CD8+ T cells among the total CD8+ T cells. Using a cutoff of 1.5%, the samples were grouped into Tex-high and Tex-low groups (Supplemental Table S2 ).
3
Single-cell Flow Cytometry Analysis
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Single-cell suspensions were prepared on ice in 2% FBS in phosphate-buffered saline (PBS). Antibody cocktails were diluted in Brilliant Violet Buffer (BD Biosciences). Samples were acquired using a BD Fortessa using FACS Diva V.6.1.3. Photomultiplier tube voltages were set using BD CS&T beads. Compensation was calculated using single-stained OneComp compensation beads (eBioscience). Samples were stained with fluorescently tagged antibodies detailed in online supplemental table 1 . Antibodies were titrated for optimal signal to noise ratio prior to use. Flow cytometry analysis was performed using FlowJo V.X and the CATALYST R package was used for FlowSOM analysis and t-distributed stochastic neighbor embedding (tSNE) projections.15 (link) All samples were gated on live, single cells.
4
Cytokine Production Assay Protocol
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Single-cell suspensions were stained at room temperature in 2% FBS in PBS. For cytokine production assays, cells were stimulated with 50 ng/mL PMA (MilliporeSigma) and 1 μg/mL ionomycin (MilliporeSigma) in the presence of GolgiPlug (BioLegend) for 4 hours. Overnight fixation was performed as needed with IC Fixation Buffer (eBioscience, Thermo Fisher Scientific). Fixation and permeabilization were performed with BD Biosciences Cytofix/Cytoperm buffers for intracellular cytokine staining. Antibody cocktails were diluted in Brilliant Violet Buffer (BD Biosciences) when using 2 or more Brilliant Violet–labeled antibodies. Samples were acquired using a BD Biosciences Fortessa operating FACSDiva 6.1.3. Photomultiplier tube voltages were set using BD Biosciences CS&T Beads. Compensation was calculated using single-stained OneComp compensation beads (eBioscience, Thermo Fisher Scientific). Samples were stained with fluorescently tagged antibodies detailed in Supplemental Table 2 . Antibodies were titrated for optimal signal-to-noise ratio prior to use. Flow cytometry analysis was performed using FlowJo v10.6. All samples were gated on single cells, lymphocytes, and CD3+CD8+ populations (Supplemental Figure 1 ). Histograms and zebra plots are used to display data.
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