Helios mass cytometer

Manufactured by Standard BioTools

Sourced in United States

The Helios mass cytometer is a high-performance instrument designed for multi-parameter single-cell analysis. It utilizes time-of-flight mass spectrometry to quantify the expression of multiple proteins simultaneously in individual cells, providing a comprehensive view of cellular phenotypes and functions.

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Helios CyTOF Mass Cytometer (14 citations) CyTOF 2 Helios upgraded mass cytometer (3 citations) Helios time-of-flight mass cytometer (2 citations) Helios time-of-flight mass cytometer (CyTOF) (2 citations) Helios model mass cytometer (2 citations) CyTOF2 Helios mass cytometer (2 citations)

140 protocols using helios mass cytometer

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).

Van Tilbeurgh M., Maisonnasse P., Palgen J.L., Tolazzi M., Aldon Y., Dereuddre-Bosquet N., Cavarelli M., Beignon A.S., Marcos-Lopez E., Gallouet A.S., Gilson E., Ozorowski G., Ward A.B., Bontjer I., McKay P.F., Shattock R.J., Scarlatti G., Sanders R.W, & Le Grand R. (2022). Innate cell markers that predict anti-HIV neutralizing antibody titers in vaccinated macaques. Cell Reports Medicine, 3(10), 100751.

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Mass Cytometry Data Analysis Protocol

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CyTOF data were acquired on a Helios mass cytometer (Fluidigm) and were analyzed using the Astrolabe Cytometry Platform (Astrolabe Diagnostics, Inc.). Single-cell data have been clustered using the FlowSOM R package (41 (link)) and labeled using the Ek’Balam algorithm (42 (link)). Cell subset definitions were followed as previously described (43 (link), 44 (link)). Cluster labeling, method implementation, and visualization were done through the Astrolabe Cytometry Platform (Astrolabe Diagnostics, Inc.).

Imanishi M., Cheng H., Kotla S., Deswal A., Le N.T., Chini E., Ko K.A., Samanthapudi V.S., Lee L.L., Herrmann J., Xu X., Reyes-Gibby C., Yeung S.C., Schadler K.L., Yusuf S.W., Liao Z., Nurieva R., Amir E.A., Burks J.K., Palaskas N.L., Cooke J.P., Lin S.H., Kobayashi M., Yoshimoto M, & Abe J.I. (2022). Radiation therapy induces immunosenescence mediated by p90RSK. Frontiers in Cardiovascular Medicine, 9, 988713.

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Multiplexed Imaging Mass Cytometry Protocol

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IMC was performed on slides dewaxed in xylene for 20 min according to previously described studies^{36 (link)}. After hydration in sequential concentrations of ethanol (100%, 95%, 80%, 70%) for 5 min, slides were incubated with antigen retrieval solution at 90–95°C for 20 min. Slides were then cooled to room temperature and washed with ddH₂O and PBS (lacking Ca⁺⁺ or Mg⁺⁺) for 5 min. After blocking with 3% BSA in PBS for 45 min, slides were labeled with metal-conjugated antibodies against CD3 (170Er - Polyclonal, C-Terminal), CD11b (149Sm - EPR1344), MHCII (174Yb - M5/114.15.2) and Ly6G (141Pr – 1A8) diluted in PBS with 0.5% BSA at 4 °C overnight. After being washed with 0.1% Triton-X in PBS and then PBS, slides were labeled with intercalator-Ir (1:2,000 dilution) in PBS (lacking Ca⁺⁺ or Mg⁺⁺) for 30 min at room temperature. After being washed again with ddH₂O for 5 min, the slides were dried. Tissues were laser ablated using a 200 Hz Hyperion™ Imaging System (Fluidigm Corp., South San Francisco, CA and the aerosol containing the ion cloud was directly transported to a Helios Mass Cytometer (Fluidigm). Images of labeled slides were obtained using the MCD viewer 1.0 (https://www.fluidigm.com/software).

Uraki R., Hastings A.K., Marin-Lopez A., Sumida T., Takahashi T., Grover J.R., Iwasaki A., Hafler D., Montgomery R.R, & Fikrig E. (2019). Aedes aegypti AgBR1 antibodies modulate early Zika virus infection of mice. Nature microbiology, 4(6), 948-955.

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High-throughput CyTOF Cytometry Protocol

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For mass cytometry (CyTOF), the 14 samples described in cohort IV were fixed, barcoded (Fluidigm), pooled into a single sample and stained with the antibody panels (Online Supplementary Table S1). Acquisition of samples was performed using a Helios mass cytometer (Fluidigm). Data were analyzed using FlowJo v.10.2 and Cytobank (Cytobank Inc.).

Majumder M.M., Leppä A.M., Hellesøy M., Dowling P., Malyutina A., Kopperud R., Bazou D., Andersson E., Parsons A., Tang J., Kallioniemi O., Mustjoki S., O’Gorman P., Wennerberg K., Porkka K., Gjertsen B.T, & Heckman C.A. (2020). Multi-parametric single cell evaluation defines distinct drug responses in healthy hematologic cells that are retained in corresponding malignant cell types. Haematologica, 105(6), 1527-1538.

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Multiparameter Analysis of Tumor Immune Landscape

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Tumors from each mouse were harvested after 10–12 days of treatment as described above. Single cell suspensions were generated from tumors using the MACS mouse tumor dissociation kit (Miltenyi Biotech Cat. 130–096-730) following manufacturer’s instructions. One million cells per tumor were resuspended in PBS and labeled with Cell-ID Cisplatin (Fluidigm, Cat. 201064) to assess for live/dead cells. For antibody labeling, we used the recommended cell surface staining procedure (Fluidigm) followed by the FoxP3/Transcription Staining Buffer Set protocol (eBiosciences™). Cells were labeled with a panel of 28 metal-conjugated antibodies to determine different immune lineages in addition to memory, trafficking, activation, and exhaustion markers (see Supplementary Table 1 for list of antibodies). After washing and centrifugation, cells were fixed using MaxPar Fix and Perm buffer (Fluidigm, Cat. 201067) and labelled for single cell discrimination with Cell-ID Intercalator-Ir (Fluidigm, Cat. 201192A). Samples were resuspended with 10% EQ four-element calibration beads (Fluidigm, Cat. 201078), and filtered through a 40 μm mesh filter prior to acquisition on a Helios™ mass cytometer (Fluidigm), at a rate of 300–500 events/s.

Márquez-Garbán D.C., Deng G., Comin-Anduix B., Garcia A.J., Xing Y., Chen H.W., Cheung-Lau G., Hamilton N., Jung M.E, & Pietras R.J. (2019). Antiestrogens in combination with immune checkpoint inhibitors in breast cancer immunotherapy. The Journal of steroid biochemistry and molecular biology, 193, 105415.

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Immunophenotyping of Bone Marrow CD34+ Cells

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BM enriched CD34 ⁺ cells were thawed and stained for viability using cisplatin protocol (Fienberg et al., 2012 (link)). After quenching and washing in CSM (at 250G for 5min), cells were stained for surface markers before fixing in 1.6% paraformaldehyde and permeabilizing in 100% methanol for 10min at 4C. GATA-1 and cleaved caspase antibodies were stained intracellularly before washing in CSM at 600G for 5min. Cells were then re-fixed in 1.6% paraformaldehyde and DNA intercalator⁴⁴ before analyzing on Helios mass cytometer (Fluidigm). Table S1 details antibodies used, their clones and the metal isotope channels they were conjugated to. Resulting FCS files from Helios run contains single cell protein level abundance for ~1mil BM cells.

Baskar R., Chen A.F., Favaro P., Reynolds W., Mueller F., Borges L., Jiang S., Park H.S., Kool E.T., Greenleaf W.J, & Bendall S.C. (2022). Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment. Cell Reports Methods, 2(3), 100188.

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Multiplexed Imaging Mass Cytometry

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Selected histospots were subjected to automated ablation using an argon-based laser in the Hyperion™ Imaging System (Fluidigm). Tissues were laser ablated in a rastering pattern at 200 Hz and the aerosol containing the ion cloud was directly transported to a Helios Mass Cytometer (Fluidigm) (16 (link), 18 (link)). The data was exported to a .txt file with counts for each metal for each pixel. TMA spots represent 350,000 – 500,000 pixels each. All raw data were analyzed using locally designed Python routines (Python Software Foundation, Wilmington, DE, USA) to study molecular distribution, marker intensity and co-localization at a pixel (1 μm²) level.

Carvajal-Hausdorf D.E., Patsenker J., Stanton K., Espindola F.V., Esch A., Montgomery R.R., Psyrri A., Kalogeras K.T., Kotoula V., Fountzilas G., Schalper K.A., Kluger Y, & Rimm D.L. (2019). Multiplexed (18-Plex) Measurement of Signaling Targets and Cytotoxic T cells in Trastuzumab-treated Patients using Imaging Mass Cytometry. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(10), 3054-3062.

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Multi-Omics Analysis of IFNγ-Treated Cells

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CMT167 and LLC cells were cultured for 48 hours under basal conditions or with 100 ng/mL IFNγ. Cells were then collected with trypsin, washed with PBS and resuspended in cisplatin (Fluidigm). Cells were incubated in cisplatin for 5 min, with the staining reaction quenched with MaxPar cell staining buffer (Fluidigm). Cells were subjected to barcoding using the Fluidigm Cell-ID 20 Plex Palladium barcoding kit according to manufacturer’s protocol (Fluidigm cat #201060). After barcoding, all samples were combined, and stained with a cocktail of metal-conjugated and fluorescently conjugated antibodies to detect cell surface proteins for 30 min at room temperature (RT) (all metal-conjugated antibodies from Fluidigm). Cells were then washed and incubated with secondary antibodies (anti-FITC, anti-PE, anti-APC, anti-Biotin) for 30 min at RT. Cells were permeablized with FoxP3 Fix/Perm buffer overnight at 4°C. Cells were then washed and incubated with antibodies to detect intracellular antigens for 2 hours at 4°C. Cells were washed, resuspended in intercalator solution, washed and resuspended with equilibration beads prior to collection on a Helios mass cytometer (Fluidigm).29 (link)

Neuwelt A.J., Kimball A.K., Johnson A.M., Arnold B.W., Bullock B.L., Kaspar R.E., Kleczko E.K., Kwak J.W., Wu M.H., Heasley L.E., Doebele R.C., Li H.Y., Nemenoff R.A, & Clambey E.T. (2020). Cancer cell-intrinsic expression of MHC II in lung cancer cell lines is actively restricted by MEK/ERK signaling and epigenetic mechanisms. Journal for Immunotherapy of Cancer, 8(1), e000441.

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Multi-parameter Mass Cytometry Profiling

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For each sample, 1 to 3 million cells were first stained with a solution containing rhodium DNA intercalator (Fluidigm) to distinguish live/dead and IdU (Fluidigm) to distinguish cells in S-phase, prior to Fc receptor blocking (Miltenyi Biotec). Samples were then stained with a mixture of metal-conjugated antibodies recognizing cell surface antigens (all antibodies purchased from Fluidigm) (SI Appendix, Table S1). After washing in Maxpar cell-staining buffer (Fluidigm), samples were fixed in ice cold methanol for 15 min prior to washing and incubation with metal-conjugated antibodies recognizing intracellular and phospho-protein antigens. Samples were washed twice in cell-staining buffer, fixed by incubation with 1.6% PFA (Pierce) for 10 min, and finally incubated overnight with iridium DNA intercalator in Maxpar fix and perm buffer (Fluidigm). Prior to acquisition, samples were washed twice in Maxpar cell-staining buffer and twice in Maxpar water and filtered through a 40-μm cell strainer before being acquired on a Helios mass cytometer (Fluidigm).
After acquisition, all .fcs files in the experiment were normalized using tools within the Helios software and then uploaded to Cytobank (https://www.cytobank.org/) for all gating and further analysis, including using clustering and dimensionality reduction algorithms such as SPADE and viSNE (85 (link)).

Cribbs A.P., Terlecki-Zaniewicz S., Philpott M., Baardman J., Ahern D., Lindow M., Obad S., Oerum H., Sampey B., Mander P.K., Penn H., Wordsworth P., Bowness P., de Winther M., Prinjha R.K., Feldmann M, & Oppermann U. (2020). Histone H3K27me3 demethylases regulate human Th17 cell development and effector functions by impacting on metabolism. Proceedings of the National Academy of Sciences of the United States of America, 117(11), 6056-6066.

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Comprehensive Immune Profiling by Mass Cytometry

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Peripheral venous blood samples were extracted into EDTA tubes in the fasting state and processed within 24 hours. PBMCs were isolated by Ficoll-Paque density gradient centrifugation and placed into a −80° C freezer until further analysis. The monoclonal antibodies used in this study are described in detail in Supplementary Table 1. According to the manufacturer’s instructions, PBMCs were stained with lanthanide metal-tagged surface antibodies, Briefly, PBMCs were washed with PBS and stained with cisplatin to assess viability. Next, PBMCs were stained with the surface markers. After surface marker staining, the cells were washed. Then, PBMCs were stained with intracellular markers. After intracellular staining, the cells were washed twice before staining in DNA intercalator solution. A Helios mass cytometer (Fluidigm, USA) was used for data acquisition.

Ge P., Liu C., Chan L., Pang Y., Li H., Zhang Q., Ye X., Wang J., Wang R., Zhang Y., Wang W., Zhang D, & Zhao J. (2022). High-Dimensional Immune Profiling by Mass Cytometry Revealed the Circulating Immune Cell Landscape in Patients With Intracranial Aneurysm. Frontiers in Immunology, 13, 922000.

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