PBMC samples were either acquired within 48 h of staining or were frozen and stored at −80°C until thawing at RT immediately prior to acquisition. For the standard Helios acquisition protocol, the samples were washed twice with PBS + 0.2% BSA, once with MilliQ H2O, and resuspended at a concentration of 750,000 cell per ml (or as indicated in the figure legends) in MiliQ H2O containing a 1/10 dilution of EQ beads (Fluidigm). These samples were acquired using a standardized acquisition template following routine tuning and instrument optimization using the narrow bore (NB) Helios injector. For the modified acquisition protocol, the samples were washed twice with PBS + 0.2% BSA, once with CAS and resuspended at a concentration of 750,000 cell per ml in CAS containing a 1/10 dilution of EQ beads (Fluidigm). These samples were acquired using a standardized acquisition template following routine tuning and optimization using the new wide-bore Helios injector after equilibrating the instrument by running CAS for at least 5 min.
Eq beads
Manufactured by Standard BioTools
EQ beads are solid-phase extraction beads used for the purification and enrichment of targeted biomolecules. They are designed to facilitate the efficient capture, washing, and elution of analytes from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Helios mass cytometer, by Standard BioTools (7 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (3 mentions)
Perm buffer, by Thermo Fisher Scientific (3 mentions)
Cisplatin, by Standard BioTools (3 mentions)
Maxpar fix and perm buffer, by Standard BioTools (3 mentions)
Cytobank software, by Beckman Coulter (2 mentions)
Cisplatin, by Enzo Life Sciences (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Cell id intercalator ir, by Standard BioTools (2 mentions)
Perm wash, by Thermo Fisher Scientific (1 mentions)
Maxpar x8 labeling kit, by Standard BioTools (1 mentions)
Helio3 cytof mass cytometer, by Standard BioTools (1 mentions)
Human trustain fcx, by BioLegend (1 mentions)
Iridium nucleic acid intercalator, by Standard BioTools (1 mentions)
Helios software, by Standard BioTools (1 mentions)
198pt monoisotopic cisplatin, by Standard BioTools (1 mentions)
Cell id 20 plex pd barcoding kit, by Standard BioTools (1 mentions)
Fc block, by BD (1 mentions)
Helios cytof mass cytometer, by Standard BioTools (1 mentions)
24 protocols using eq beads
1
Standardized PBMC Acquisition Protocols
2
Cryopreserved Whole Blood CyTOF Protocol
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Whole blood was collected in heparin/lithium tubes and cryopreserved in 10% FCS/DMSO. Samples were rapidly thawed, washed with RPMI medium, and then incubated in RPMI supplemented with 10 mg/mL DNAse for 30 min at 37°C. Cells were then resuspended in 1× PBS and incubated for 15 min at 37°C with 1 μL Intercalator-Rh (DVS Sciences). After washing in PBS-0.5% BSA, cells were stained for 30 min at 4°C, washed with 1× PBS, fixed with PBS-1.6% PFA, and washed with 1× Perm/Wash Buffer (eBiosciences). After permeabilization with 1× Perm/Wash (eBiosciences), intracellular staining was performed for 30 min at 4°C. Cells were washed, fixed in 1.6% PFA, and washed with Barcode Perm Buffer (DVS) before barcoding with a unique combination of three palladium isotopes (DVS, Fluidigm) according to the manufacturer’s recommendations. Cells were then washed with PBS and resuspended in PBS-1.6% PFA containing 0.5 μL Intercalator-Ir (DVS Sciences). Barcoded samples were pooled and stored overnight at 4°C. Before acquisition, cells were washed with milli-Q water and filtered through a cap filter with 35-μm pores (BD Biosciences). Normalization beads (Eq Beads, Fluidigm) were added to the tube and the acquisition was performed using a Helios mass cytometer (Fluidigm). Data were acquired for six consecutive days (3 animals per day).
3
Single-Cell Mass Cytometry Analysis
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A summary of the 42 mass-conjugated antibodies used in the study to identify immune cells is shown in Supplementary Table 2 . For mass cytometry acquisition, cells were resuspended in ddH20 containing EQ beads (Fluidigm). All samples were assayed on a Helio3 CyTOF mass flow cytometer (Fluidigm), and the data obtained were normalized using the bead-based normalizer [21 (link)]. CyTOF analysis was conducted by PLTTech Inc. (Hangzhou, China) following the previously stated protocol [22 (link)]. All 42 immune cell markers were used to cluster and visualize. Cells were clustered based on the X-shift algorithm [23 (link)]. 100,000 cells per sample were randomly selected for clustering, and the cells with less than 100,000 were all input. All marker expressions (raw data) were arcsinH-transformed with a cofactor of 5 (counts_transf = asinh(x/5)). For visualization of high-dimensional data on two dimensions, we analyzed 10,000 randomly selected cells from each sample using the R package cytofkit by the t-SNE algorithm [24 (link)]. Immune cell subpopulations were identified based on the median value of specific markers expression in the hierarchical clusters. For marker expression level visualization on t-SNE plots, the expression was normalized.
4
Mass Cytometry Data Analysis Protocol
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The open source statistical package R (https://www.r-project.org/ ) was used for all statistical analyses [41 ]. Mass cytometry data were bead-normalized with EQ Beads (Fluidigm) prior to subsequent analyses, using the Premessa package (https://github.com/ParkerICI/premessa ) [42 (link)]. Data were first visualized with FlowJo v10.5.3 (Tree Star, Woodburn, OR, USA). Markers noted to have poor staining were excluded from subsequent analyses (FAS-L, Ki-67, KIR2DS2 and CXCR6 for Panel 1; CD11a and KIR2DS2 for Panel 2). Serial negative gating was used to identify NK cells, as described [40 (link)] (S1 Fig ). Normalized signal intensities were transformed using the inverse hyperbolic sine (asinh) function with a cofactor equal to 5 to account for heteroskedasticity, prior to subsequent analyses. The data supporting this publication is available at ImmPort (https://www.immport.org ) under study accession SDY1647.
5
CyTOF Data Normalization and Analysis
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Data analysis was performed using the workflow previously developed (23 (link)). Briefly, after acquisition, intrafile signal drift was normalized and.fcs files were obtained using CyTOF software. To diminish batch effects, all files were normalized on EQ Beads (Fluidigm) using the premessa R package (https://github.com/ParkerICI/premessa ). Raw median intensity values were transformed to a hyperbolic arcsine (arcsinh) and then analysis was performed using Cytobank software (Beckman Coulter, Brea, CA, USA).
6
Mass Cytometry Cell Staining Protocol
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All antibodies were conjugated using MaxPar X8 labeling kits (Fluidigm), except for those purchased directly from Fluidigm; details of all antibodies is given in Supplementary Table S1 . To maintain antibody stability and consistency in staining, all antibody panels were pre-mixed into separate surface and ICS cocktails (as indicated in Supplementary Table S1 ), aliquoted and frozen at −80°C until use. Palladium (Pd102, Pd104, Pd106, and Pd108) conjugated CD45 antibodies for barcoding were made as previously described (28 (link)).
At the end of co-culture, cells were stained for viability using 25 μM Cisplatin (Enzo) for 1 min and quenched with FBS, and samples were barcoded using palladium-based CD45 barcodes as previously described (28 (link)). After barcoding, cells were washed thrice, and all samples from a set of barcodes were combined. Samples were stained with the surface antibody panel for 30 min at 4°C, fixed with 2% paraformaldehyde (PFA, Electron Microscopy Sciences), permeabilized with Permeabilization Buffer (eBioscience), and stained with the intracellular staining (ICS) panel (made in Permeabilization Buffer) for 45 min at 4°C. Cells were suspended overnight in iridium interchelator (DVS Sciences) in 2% PFA, and resuspended in 1x EQ Beads (Fluidigm) before acquisition on a Helios mass cytometer (Fluidigm).
At the end of co-culture, cells were stained for viability using 25 μM Cisplatin (Enzo) for 1 min and quenched with FBS, and samples were barcoded using palladium-based CD45 barcodes as previously described (28 (link)). After barcoding, cells were washed thrice, and all samples from a set of barcodes were combined. Samples were stained with the surface antibody panel for 30 min at 4°C, fixed with 2% paraformaldehyde (PFA, Electron Microscopy Sciences), permeabilized with Permeabilization Buffer (eBioscience), and stained with the intracellular staining (ICS) panel (made in Permeabilization Buffer) for 45 min at 4°C. Cells were suspended overnight in iridium interchelator (DVS Sciences) in 2% PFA, and resuspended in 1x EQ Beads (Fluidigm) before acquisition on a Helios mass cytometer (Fluidigm).
7
CyTOF Antibody Profiling Protocol
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Table S1
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8
Mass Cytometry: Streamlined Cell Analysis
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Mass cytometry staining and data acquisition were performed as previously described (11 (link)). See Supplemental Table 3 for antibody panel information. In brief, PBMCs were thawed and washed with FACS buffer. Then 4 × 106 or fewer cells per patient were stained with live/dead (1 μM 198PT monoisotopic cisplatin; Fluidigm). Cells were incubated in Cytofix fixation buffer, washed, and barcoded using palladium metal barcodes as per the manufacturer’s instructions (Fluidigm). Cells were incubated with Human TruStain FcX (BioLegend) and stained with an antibody master mix for 30 minutes at room temperature. After washing, cells were fixed with 2.4% formaldehyde in PBS containing 125 nM iridium nucleic acid intercalator (Fluidigm) and kept overnight. Cells were cryopreserved and stored at –80°C until thawing for acquisition. Cells were washed and resuspended at a concentration of 1 × 106 cells/mL in cell acquisition solution with 5% EQ beads (Fluidigm). Acquisition was performed using a Helios mass cytometer (Fluidigm) and a standardized acquisition template. FCS files were bead-normalized and debarcoded using Helios software (Fluidigm). Using FlowJo (BD), debris, dead cells, and doublets were excluded.
9
Comprehensive PBMC Phenotyping by Mass Cytometry
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Cryopreserved PBMC were thawed by incubating cryovials at 37°C for 1 min and stained with the viability marker Cisplatin. Subsequently, PBMCs were incubated with an antibody mixture for 30 min. After washing, PBMCs were fixed in PBS (Thermo Fisher) with 2% PFA (Sigma-Aldrich) overnight at 4°C. The next day, PBMCs were stained with an intracellular antibody mixture after permeabilization using saponin-based Perm Buffer (eBioscience). After washing, cellular DNA was labeled with Cell-ID Intercalator-Ir (Fluidigm) and cell pellets were resuspended in 1:10 EQ Beads (Fluidigm) in 1 mL MiliQ water. Samples were measured using a Helios mass cytometer (Fluidigm). Twenty One different PBMC cell subsets were identified using the unsupervised gating approach DAFi60 (link) with the exception of antibody-secreting cells (ASCs), which were manually gated as CD45+Live+CD14−CD3−CD19+CD20−CD38+ cells. Gating was performed using FlowJo (BD, version 10.7.0).
10
Multiparameter Mass Cytometry Profiling
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