5x106 peripheral blood mononuclear cells (PBMCs) were stained with Cell-ID Cisplatin-195Pt (Fluidigm, South San Francisco, CA) to discriminate dead cells. PBMCs were then FcR blocked with Human TruStain FcX (Biolegend, San Diego, CA), stained with the antibody mix as shown in Table S2
Helios cytof 3
Manufactured by Standard BioTools
The Helios CyTOF 3.0 is a high-dimensional, single-cell analysis instrument developed by Standard BioTools. It utilizes mass cytometry technology to enable the simultaneous measurement of over 40 parameters per single cell. The Helios CyTOF 3.0 provides researchers with a powerful tool for in-depth cellular analysis and characterization.
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3 protocols using helios cytof 3
1
Comprehensive PBMC Immunophenotyping by CyTOF
2
Multiparametric Characterization of PBMCs by CyTOF
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Approximately 5 × 10 6 PBMCs were stained with Cell-ID Cisplatin-195Pt to discriminate dead cells. PBMCs were then FcR blocked with Human TruStain FcX (Biolegend #422302), stained with the antibody mix as shown in Supplementary Table S1, fixed with 1.6% paraformaldehyde, and stained with Cell-ID intercalator-Ir. Stained PBMCs were analyzed in a Helios CyTOF 3.0 (Fluidigm) following the manufacturer's instructions at the Cancer and Immunology Core at Vanderbilt University. The data were exported as an FCS and normalized using EQ-Four calibration beads standards (Fluidigm # 201078) following the manufacturer's protocol. For multidimensional analysis, the data were pre-gated to remove dead cells and debris, then gated for CD45 + cells using FlowJo 10.7.2 (Becton, Dickinson & Company). The pre-gated data were exported as FCS files and then imported into RStudio. We used the package CATALYST [7] (link) to arcsine transform marker intensities with a cofactor of five and performed subsequent analysis. Unsupervised clustering was performed using FlowSOM [8] (link), and data representation was performed using the R package ggplot2. Manual gating was performed in FlowJo. Frequencies of various populations were exported from FlowJo and analyzed using Prism 9 (GraphPad Software).
3
CyTOF Phenotyping of PBMC
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Staining and data acquisition: 5x10 6 peripheral blood mononuclear cells (PBMCs) were stained with Cell-ID Cisplatin-195 Pt (Fluidigm, South San Francisco, CA) to discriminate dead cells. PBMCs were then FcR blocked with Human TruStain FcX (Biolegend, San Diego, CA), stained with the antibody mix as shown in table S1, xed with 1.6% paraformaldehyde, and stained with Cell-ID intercalator-Ir. Stained PBMCs were analyzed in a Helios CyTOF 3.0 (Fluidigm) following manufacturer's instructions at the Cancer and Immunology Core at Vanderbilt University. The data was exported as a Flow Cytometry Standard le (FCS) and normalized using EQ bead standards (Fluidigm) following manufacturer's protocol.
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