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Helios cytof mass cytometer

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The Helios CyTOF Mass Cytometer is a laboratory instrument designed for high-dimensional single-cell analysis. It utilizes mass cytometry technology to detect and quantify multiple protein and cellular markers simultaneously within individual cells. The core function of the Helios CyTOF Mass Cytometer is to enable researchers to obtain comprehensive, high-dimensional data from complex biological samples.

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Lab products found in correlation

Eq four element calibration beads, by Standard BioTools (3 mentions) Cell acquisition solution, by Standard BioTools (3 mentions) Iridium intercalator solution, by Standard BioTools (2 mentions) Cell id cisplatin, by Standard BioTools (2 mentions) Lyve1, by BD (1 mentions) Golgiplug golgistop, by BD (1 mentions) Cd103, by BD (1 mentions) Cd163, by BD (1 mentions) Anti nox2, by BD (1 mentions) Bromodeoxyuridine (brdu), by BD (1 mentions) Siglec f, by BD (1 mentions) 70 m cell strainer, by BD (1 mentions) Maxpar reagent kits, by Standard BioTools (1 mentions) Benzonase nuclease, by Merck Group (1 mentions) Trustain fcx, by BioLegend (1 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions) Eq calibration beads, by Standard BioTools (1 mentions) Human fc receptor blocking solution, by BioLegend (1 mentions) Maxpal cell staining buffer, by Standard BioTools (1 mentions) Maxpal fix and pem buffer, by Standard BioTools (1 mentions)

14 protocols using helios cytof mass cytometer

1

Multiparameter CyTOF Analysis of PBMCs

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PBMCs were stained with 37 metal-conjugated antibodies (Lymphoid panel, Supplementary Table S3) as previously described (15 (link), 17 (link)). After staining, data acquisition was done on the Helios CyTOF mass cytometer (Fluidigm). The generated files were analyzed using FlowJo v10.5.1 (FlowJo, USA) and down-sampled to equal number of live immune cell events of 12906 (smallest possible events of the 13 files) for comparison. To identify cell populations, we clustered the merged data containing 167778 single cell events using the FlowSOM (18 (link)) method. Clusters showing significant temporal frequency changes were detected using repeated measures ANOVA, where linear mixed models were used to account for missing data (19 ). Clusters were assigned to 7 major cell lineages and visualized with t-stochastic neighbor embedding (t-SNE) (20 (link)). Clustering results were validated by manual gating using FlowJo v10.5.1. Statistical analysis and data visualization were performed with R packages (21 ). In another CyTOF experiment to investigate MDSC, PBMC were stained with metal-conjugated antibodies (Myeloid panel, Supplementary Table S3) as described above. The CyTOF data were manually gated to check for the frequencies of MDSC using FlowJo.
Lim C.J., Nguyen P.H., Wasser M., Kumar P., Lee Y.H., Nasir N.J., Chua C., Lai L., Hazirah S.N., Loh J.J., Khor L.Y., Yeong J., Lim T.K., Low A.W., Albani S., Chong T.W, & Chew V. (2021). Immunological Hallmarks for Clinical Response to BCG in Bladder Cancer. Frontiers in Immunology, 11, 615091.
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2

PBMC Acquisition using CyTOF Mass Cytometry

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Before acquisition, PBMCs were washed twice with CSB and resuspended at a concentration of 1.1×106 cells/mL in the Cell Acquisition Solution (Fluidigm) containing 10% EQ Four Element Calibration Beads (Fluidigm). PBMCs were acquired using a Helios CyTOF Mass Cytometer (Fluidigm) equipped with a SuperSampler fluidics system (Victorian Airships), and data were collected as previously described. fcs files.
He X., Cui X., Zhao Z., Wu R., Zhang Q., Xue L., Zhang H., Ge Q, & Leng Y. (2024). A generalizable and easy-to-use COVID-19 stratification model for the next pandemic via immune-phenotyping and machine learning. Frontiers in Immunology, 15, 1372539.
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3

Comprehensive Myeloid Cell Profiling by CyTOF

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CyTOF allows for high-dimensional analysis of cell surface markers, cytokines and signaling molecules simultaneously at the single-cell level20 (link). Thus, we used the CyTOF for phenotyping and function assay for the mouse lung myeloid populations including IMs, AMs, monocytes, neutrophils, DCs and eosinophils. A panel of metal-labelled antibodies (Supplementary Table 4) including several markers for macrophages and myeloid cells as well as cytokines and signaling molecules (CD45, CD11b, F4/80, CD64, Ly6C, Ly6G, MerTK, CD24, CD206, SiglecF, CD11c, CD301, Arginase 1, LGR4, β-Catenin, IL-10, iNOS, TNF, CX3CR1, CD80, CD69, CD86, CCR2, CD115, I-A/I-E, BST2, RELMα, CD103, CD3, CD49, TER-119) were used for staining according the FLUIDIGM Inc recommended protocol. Samples were run on the Helios CyTOF Mass cytometer (FLUIDIGM Inc, USA) at the flow cytometry core of Research Resources Center of the University of Illinois at Chicago. Data from the CyTOF were analyzed using Cytobank online analysis tool (www.cytobank.org/, Cytobank Inc, USA).
Zhou B., Magana L., Hong Z., Huang L.S., Chakraborty S., Tsukasaki Y., Huang C., Wang L., Di A., Ganesh B., Gao X., Rehman J, & Malik A.B. (2020). The angiocrine Rspondin3 instructs interstitial macrophage transition via metabolic-epigenetic reprogramming and resolves inflammatory injury. Nature immunology, 21(11), 1430-1443.
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4

High-Dimensional CyTOF Analysis of Cell Markers

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We used the CyTOF for high-dimensional analysis of cell surface markers, cytokines, and signaling molecules simultaneously at the single-cell level. A panel of 37 metal-labeled antibodies (CD45, CD16/32, CD64, CD11b, CD11c, CD206, CD80, CD86, MHC-II (I-A/I-E), MHC-I, CX3CR1, F4/80, CD169, Ly6G, Ly6C, CD19, CD4, CD8, NK1.1, Epcam, TNFα, IL-10, and active Caspase-3 were purchased from FLUDIGM (Table S2). Primary antibodies for SiglecF, MERTK, Tim4, Marco, CD24, Arginase-1 (Arg1), CCR2, CD68, V-ATPase, Anti-NOX2, Lyve1, CD163, CD31, CD103, and BrdU were purchased from BD Bioscience, R&D Biosystems, BioLegend, and Thermo Fischer (Table S2), and labeled with FLUDIGM metal labeling kit (Maxpar X8 metal Labeling Kit) according to the manufacturer’s protocol. Cells were treated with Golgistop/GolgiPlug (BD Bioscience) for 2–3 h at 37°C and stained according to the FLUIDIGM recommended protocol. Samples were run on the Helios CyTOF mass cytometer (FLUIDIGM) at the flow cytometry core of the Research Resources Center of the University of Illinois at Chicago. Data from CyTOF were analyzed using the Cytobank online analysis tool (https://www.cytobank.org).
Chakraborty S., Singh A., Wang L., Wang X., Sanborn M.A., Ye Z., Maienschein-Cline M., Mukhopadhyay A., Ganesh B.B., Malik A.B, & Rehman J. (2023). Trained immunity of alveolar macrophages enhances injury resolution via KLF4-MERTK-mediated efferocytosis. The Journal of Experimental Medicine, 220(11), e20221388.
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5

CyTOF Analysis of Tumor Microenvironment

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Tumors were digested in a PBS‐based buffer (with 2% FBS) that contained both collagenase and DNAse. Subsequently, the cell homogenates were filtered through 70‐µm cell strainers (BD Biosciences) and immediately treated with CyTOF antibodies. For staining of cells, the cells were washed with 1× PBS, centrifuged at 300 × g, fixed with 1.6% formaldehyde, and then exposed to cisplatin viability staining reagent (Fluidigm) for ≈3 min. The cells were incubated in the antibody suspension for 30 min. The antibodies used for the CyTOF staining panel are listed in Table2. DNA was stained by adding an iridium intercalator solution (Fluidigm) to the cells. The cells were subsequently washed and reconstituted in Maxpar Cell Acquisition Solution with EQ four‐element calibration beads (Fluidigm) and then analyzed on a Helios CyTOF Mass Cytometer (Fluidigm).
CyTOF data were initially normalized using Normalizer and Debarcoding software. Data analysis was performed using the FlowJo v10 Software (BD Biosciences), Cytobank (Mountain View, CA), and the viSNE and CITRUS algorithms, enabling the mapping of single‐cell data in two dimensions as a tSNE plot. The tSNE plots were then used to understand the change in intratumoral microenvironment.
Bi J., Witt E., Voltarelli V.A., Feig V.R., Venkatachalam V., Boyce H., McGovern M., Gutierrez W.R., Rytlewski J.D., Bowman K.R., Rhodes A.C., Cook A.N., Muller B.N., Smith M.G., Ramos A.R., Panchal H., Dodd R.D., Henry M.D., Mailloux A., Traverso G., Otterbein L.E, & Byrne J.D. (2023). Low‐Cost, High‐Pressure‐Synthesized Oxygen‐Entrapping Materials to Improve Treatment of Solid Tumors. Advanced Science, 10(10), 2205995.
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6

CyTOF Staining of Thawed Cells

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Cells were thawed for 3 minutes at 37°C in culture medium. 20 U/mL heparin sodium salt and 25 U/mL benzonase nuclease were added to prevent cell clumping (Sigma-Aldrich, St. Louis, MO). CyTOF staining was performed at room temperature. Cells were stained with Cell-ID cisplatin (Fluidigm, San Francisco, CA) for viability, washed, and blocked with TruStain FcX (BioLegend, San Diego, CA) for 10 minutes. Cells were then stained with CyTOF antibodies labeled with rare earth metal isotopes using MaxPar reagent kits from Fluidigm (Table S2, Supplemental Digital Content 1) for 30 minutes. After fixation and permeabilization of the cells using the FoxP3 Staining Buffer Set (eBioscience, San Diego, CA), samples were barcoded using combinations of palladium isotopes as described (18 (link)), allowing the entire mixture of samples to be analyzed in a small number of CyTOF runs over 2 days. 18.75 μM iridium intercalator solution (Fluidigm, San Francisco, CA) was added to the cells, and cells were subsequently washed and reconstituted in Milli-Q filtered distilled water with EQ four element calibration beads (Fluidigm, San Francisco, CA). Cells were analyzed on a Helios CyTOF Mass Cytometer (Fluidigm, San Francisco, CA) (19 (link)).
Riça I.G., Joughin B.A., Teke M.E., Emmons T.R., Griffith A., Cahill L.A., Banner-Goodspeed V., Robson S.C., Hernandez J.M., Segal B.H., Otterbein L.E., Hauser C.J., Lederer J.A, & Yaffe M.B. (2022). Neutrophil heterogeneity and emergence of a distinct population of CD11b/CD18- activated low-density neutrophils after trauma. The journal of trauma and acute care surgery, 94(2), 187-196.
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7

CyTOF Cellular Staining and Analysis

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Metal-conjugated antibodies used for CyTOF analyses are presented in Key resource table. The CyTOF assay was performed according to the manufacturer’s instruction. Briefly, 3 × 106 cells were stained with 5mM Cell-ID Cisplatin (Fluidigm, San Francisco, CA) for 5 minutes and quenched with MaxPal Cell Staining Buffer (Fluidigm) using 5 times the volume of the cell suspension. After centrifugation, cell suspensions (50 μl) were incubated with 5 μl of human Fc-receptor blocking solution (Biolegend, San Diego, CA) for 10 minutes and 50 μl of pre-mixed antibody cocktail for 30 minutes. After washing, cells were incubated with 1 ml of cell intercalation solution (125nM MaxPal Intercalator-Ir into 1 ml MaxPal Fix and Pem Buffer, Fluidigm) overnight at 4°C. Cells were centrifuged with MaxPal Water and pelleted. The pelleted cells were suspended with EQ Calibration Beads (Fluidigm) and cell events were acquired by a Helios CyTOF mass cytometer (Fluidigm).
Hailemichael Y., Johnson D.H., Abdel-Wahab N., Foo W.C., Bentebibel S.E., Daher M., Haymaker C., Wani K., Saberian C., Ogata D., Kim S.T., Nurieva R., Lazar A.J., Abu-Sbeih H., Fa-ak F., Matthew A., Wang Y., Falohun A., Trinh V., Zobniw C., Spillson C., Burks J.K., Awiwi M., Elsayes K., Soto L.S., Melendez B.D., Davies M., Wargo J., Curry J., Yee C., Lizee G.A., Singh S., Sharma P., Allison J.P., Hwu P., Ekmekcioglu S, & Diab A. (2022). Interleukin-6 blockade abrogates immunotherapy toxicity and promotes tumor immunity. Cancer cell, 40(5), 509-523.e6.
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8

Murine Splenic Cell Characterization via CyTOF

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Spleens were harvested from mice and the tissue was homogenized. Cells were washed twice with PBS and counted with the ViCell XR analyzer and resuspended in wash buffer (0.5% BSA in PBS). A cell surface antibody mix was added to each sample and put on the shaker for 1 h at room temperature. Cells were then washed three times to remove residual antibodies and 1 μM Pt198 (Fluidigm), a live/dead stain, was added to each sample for 5 min on the shaker at room temperature. Cells were washed three times, then 4% paraformaldehyde was added to each sample and were incubated for 15 min at room temperature. Cells were washed once, then 1 mL 100% methanol was added to each sample overnight. After one wash, intracellular antibody mix was added to each sample with incubation for 1 h on the shaker at room temperature. Cells were washed two times, then Ir‐intercalator‐193, 125 μM (Fluidigm, California, USA) was added to each sample with a final dilution of 1:2000. Samples were analyzed with the Fluidigm Helios CYTOF Mass Cytometer.
Rodriguez M., Fekry B., Murphy B., Figueroa M., Cheng T., Raber M., Wartenberg L., Bell D., Triche L., Crawford K., Ma H., Allton K., Ahmed R., Tran J., Ranieri C., Konopleva M., Barton M., Nunez C., Eckel‐Mahan K, & Chandra J. (2024). Feasible diet and circadian interventions reduce in vivo progression of FLT3‐ITD‐positive acute myeloid leukemia. Cancer Medicine, 13(2), e6949.
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9

PBMC Acquisition and CyTOF Analysis

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Before acquisition, PBMCs were washed twice by CSB and then resuspended at a concentration of 1.1 × 106 cells/mL in Cell Acquisition Solution (Fluidigm) containing 10% of EQ Four Element Calibration Beads (Fluidigm). The PBMCs were acquired on the Helios CyTOF Mass Cytometer (Fluidigm) equipped with a SuperSampler fluidics system (Victorian Airships), and data were collected as .fcs files. CyTOF analyses were performed by National Research Center for Translational Medicine at Shanghai.
Yu S., Di C., Chen S., Guo M., Yan J., Zhu Z., Liu L., Feng R., Xie Y., Zhang R., Chen J., Wang M., Wei D., Fang H., Yin T., Huang J., Chen S., Lu H., Zhu J, & Qu J. (2021). Distinct immune signatures discriminate between asymptomatic and presymptomatic SARS-CoV-2pos subjects. Cell Research, 31(11), 1148-1162.
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10

High-Dimensional Immune Profiling of BMMCs

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BMMCs were thawed, washed, and barcoded according to manufacturer recommendations (Fluidigm). Barcoded cells were stained using a custom 39-marker panel designed to characterize innate and adaptative immune cell subtypes (online supplemental figure 1). Stained cells were acquired on a CyTOF Helios mass cytometer (Fluidigm). Automated cell classification was performed using the Astrolabe Mass Cytometry Platform (Astrolabe Diagnostics, Inc.). Briefly, immune subsets were clustered into self-organizing maps using the FlowSOM package and labeled using the Ek’Balam algorithm.15 (link) Cell subset definitions follow previously reported definitions of the healthy human immune system and the Human ImmunoPhenotyping Consortium.16 (link) Cell types were excluded if there were less than three cells in at least half of all samples. Plasmablasts, which primarily represent tumor cells, were excluded from the calculation of immune cell frequency.17 18 (link)
Coffey D.G., Osman K., Aleman A., Bekri S., Kats S., Dhadwal A., Catamero D., Kim-Schulze S., Gnjatic S., Chari A., Parekh S., Jagannath S, & Cho H.J. (2024). Phase 1 study combining elotuzumab with autologous stem cell transplant and lenalidomide for multiple myeloma. Journal for Immunotherapy of Cancer, 12(4), e008110.
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