Helios instrument

Manufactured by Standard BioTools

The Helios instrument is a high-resolution mass spectrometer designed for advanced analytical applications. It provides accurate and precise mass measurements, enabling detailed analysis of complex samples.

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12 protocols using helios instrument

Mass Cytometry Analysis of Immune Cells in Arthritis

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We collected 6 leukocyte-rich, 9 leukocyte-poor RA, and 11 OA samples for mass cytometry analysis, and processed the samples, as described previously^{22 (link)}. Briefly, we analyzed samples on a Helios instrument (Fluidigm) after antibody staining and fixation (Supplementary Table 2). Mass cytometry data were normalized using EQ™ Four Element Calibration Beads (Fluidigm), as previously described^{52 (link)}. Cells were first gated to live DNA+ cells prior to gating for specific cell populations using the following scheme: B cells (CD3⁻CD14⁻CD19⁺), fibroblasts (CD45⁻PDPN⁺), monocytes (CD3⁻CD14⁺), and T cells (CD3⁺CD14⁻). All biaxial gating was performed using FlowJo 10.0.7.

Zhang F., Wei K., Slowikowski K., Fonseka C.Y., Rao D.A., Kelly S., Goodman S.M., Tabechian D., Hughes L.B., Salomon-Escoto K., Watts G.F., Jonsson A.H., Rangel-Moreno J., Pellett N.M., Rozo C., Apruzzese W., Eisenhaure T.M., Lieb D.J., Boyle D.L., Mandelin AM I.I., Boyce B.F., DiCarlo E., Gravallese E.M., Gregersen P.K., Moreland L., Firestein G.S., Hacohen N., Nusbaum C., Lederer J.A., Perlman H., Pitzalis C., Filer A., Holers V.M., Bykerk V.P., Donlin L.T., Anolik J.H., Brenner M.B., Raychaudhuri S., Albrecht J., Bridges SL J.r., Buckley C.D., Buckner J.H., Dolan J., Guthridge J.M., Gutierrez-Arcelus M., Ivashkiv L.B., James E.A., James J.A., Keegan J., Lee Y.C., McGeachy M.J., McNamara M.A., Mears J.R., Mizoguchi F., Nguyen J.P., Noma A., Orange D.E., Rohani-Pichavant M., Ritchlin C., Robinson W.H., Seshadri A., Sutherby D., Seifert J., Turner J.D, & Utz P.J. (2019). Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry. Nature immunology, 20(7), 928-942.

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Multiparametric Profiling of PBMCs

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PBMCs were thawed as previously described ^{29 (link)}, then processed and stained according to the Fluidigm PRD017 V3 protocol, as discussed below. First, cells were washed and incubated with cisplatin (final concentration 5μM) for dead cells exclusion. Next, cells were blocked with 0.5mg/mL Human Fc Block (Biolegend) 10 min at room temperature (RT) and stained 30 min at RT with 27 extracellular antibodies (Supplementary Table 1). Then, cells were fixed (2% PFA, 30 min RT), permeabilized with Barcode Perm Buffer (Fluidigm), and barcoded using the Cell-ID™ 20-Plex Pd Barcoding Kit (Fluidigm). Afterwards, they were incubated with Human Fc Block and stained 30 min at RT with 4 intracellular antibodies (Supplemental Table 1) and labeled overnight with 125nM iridium intercalator (Fluidigm). Finally, cells were diluted in EQTM Four Element Calibration Beads (Fluidigm) before acquisition on a Helios^® instrument (Fluidigm).

Freeman R., Magee J., Barratt A., Wheeler J., Steward M., Lee M, & Piggott N. (1999). Rapid Immunochromatographic Assay for Diagnosis of Tuberculosis: Antibodies Detected May Not Be Specific. Journal of Clinical Microbiology, 37(6), 2111-2112.

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High-Dimensional Profiling of Immune Cells by CyTOF

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CyTOF staining and analysis were performed as described^{41 (link)}. The antibodies used in the CyTOF analyses are summarized in Supplementary Table 2. Cells were subjected to staining after washing with PBS supplemented with 2% fetal calf serum (FCS, Biosera, Orange, CA, USA) (washing solution). The cells were incubated in 5 μM of Cell-ID rhodium solution (Fluidigm, South San Francisco, CA) in PBS, washed using washing solution, and stained with a mixture of surface-staining antibodies (1:100 dilution). After washing, the cells were fixed and permeabilized using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. The fixed and permeabilized cells were stained with the intracellular antibodies (1:50 dilution). After washing twice, the cells were allowed to rest overnight in 125 nM MaxPar Intercalator-Ir (Fluidigm) diluted in PBS solution with 2% paraformaldehyde at 4°C. The cells were then washed once with washing solution and twice with MaxPar water (Fluidigm) and distilled water with minimal heavy element contamination to reduce the background level. The cells were resuspended in MaxPar water supplemented with 10% EQ Four Element Calibration Beads (Fluidigm) and then were applied to the Helios instrument (Fluidigm), and data were acquired at a speed below 300 events/second.

Maeda Y., Wada H., Sugiyama D., Saito T., Irie T., Itahashi K., Minoura K., Suzuki S., Kojima T., Kakimi K., Nakajima J., Funakoshi T., Iida S., Oka M., Shimamura T., Doi T., Doki Y., Nakayama E., Ueda R, & Nishikawa H. (2021). Depletion of central memory CD8+ T cells might impede the antitumor therapeutic effect of Mogamulizumab. Nature Communications, 12, 7280.

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Synovial Cell Immunophenotyping by CyTOF

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Synovial cells were resuspended in PBS/1%BSA with primary antibody cocktails at 1:100 dilution for 30 min (Additional file 1: Table S1). All antibodies were obtained from the Longwood Medical Area CyTOF Antibody Resource Core (Boston, MA, USA). Cisplatin was added at 1:400 dilution for the last 5 min of the stain to assess viability. Cells were then washed and fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were then washed and incubated with Ebioscience Transcription Factor Fix/Perm Buffer for 30 min, washed in Ebioscience perm buffer, and stained for intracellular markers at 1:100 for 30 min. Cells were refixed in 1.6% paraformaldehyde for 10 min and stored overnight in PBS/1%BSA. The following day, cells were incubated with MaxPar Intercalator-Ir 500 μM 1:4000 in PBS for 20 min, and then washed twice with MilliQ water, filtered, and analyzed on a Helios instrument (Fluidigm). Mass cytometry data were normalized using EQ™ Four Element Calibration Beads (Fluidigm) as described previously [21 (link)]. viSNE analysis was performed using the Barnes-Hut SNE implementation on Cytobank (www.cytobank.org). Gated live cells (DNA-positive, cisplatin-negative) were analyzed using all available protein markers. Biaxial gating was performed using FlowJo 10.0.7.

Donlin L.T., Rao D.A., Wei K., Slowikowski K., McGeachy M.J., Turner J.D., Meednu N., Mizoguchi F., Gutierrez-Arcelus M., Lieb D.J., Keegan J., Muskat K., Hillman J., Rozo C., Ricker E., Eisenhaure T.M., Li S., Browne E.P., Chicoine A., Sutherby D., Noma A., Nusbaum C., Kelly S., Pernis A.B., Ivashkiv L.B., Goodman S.M., Robinson W.H., Utz P.J., Lederer J.A., Gravallese E.M., Boyce B.F., Hacohen N., Pitzalis C., Gregersen P.K., Firestein G.S., Raychaudhuri S., Moreland L.W., Holers V.M., Bykerk V.P., Filer A., Boyle D.L., Brenner M.B, & Anolik J.H. (2018). Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue. Arthritis Research & Therapy, 20, 139.

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Characterizing Immune Cell Populations in Tumor and Blood

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Peripheral blood mononuclear cells and tumour cells were collected and washed twice with wash buffer (0.5% bovine serum albumin (BSA) in PBS). For tumour, this included 9 responders and 9 non-responders, and for peripheral blood mononuclear cells, 8 responders and 8 non-responders. To determine the live population, cells were stained with 1 μM cisplatin for 3 min. The reaction was stopped with FACS buffer (2% fetal bovine serum (FBS) in PBS), and the cells were washed once with wash buffer. Cells were then incubated with 5 μl of Fc receptor blocking buffer reagent (Miltenyi) for 10 min at room temperature. Cells were incubated with surface antibodies at room temperature for 60 min, washed twice with wash buffer and stored overnight in 1 ml of 1.6% paraformaldehyde (EMD Biosciences) in PBS with 125 nM iridium nucleic acid intercalator (Fluidigm). The next day, samples were washed twice with cell staining buffer, re-suspended in 1 ml of MilliQ dH2O, filtered through a 35-μm nylon mesh (cell strainer cap tubes, BD) and counted. Before analysis, samples were resuspended in MilliQ dH₂O supplemented with EQ four element calibration beads at a concentration of 0.5 × 10⁵ per ml. Samples were acquired at 300 events per second on a Helios instrument (Fluidigm) using the Helios 6.5.358 acquisition software (Fluidigm).

Helmink B.A., Reddy S.M., Gao J., Zhang S., Basar R., Thakur R., Yizhak K., Sade-Feldman M., Blando J., Han G., Gopalakrishnan V., Xi Y., Zhao H., Amaria R.N., Tawbi H.A., Cogdill A.P., Liu W., LeBleu V.S., Kugeratski F.G., Patel S., Davies M.A., Hwu P., Lee J.E., Gershenwald J.E., Lucci A., Arora R., Woodman S., Keung E.Z., Gaudreau P.O., Reuben A., Spencer C.N., Burton E.M., Haydu L.E., Lazar A.J., Zapassodi R., Hudgens C.W., Ledesma D.A., Ong S., Bailey M., Warren S., Rao D., Krijgsman O., Rozeman E.A., Peeper D., Blank C.U., Schumacher T.N., Butterfield L.H., Zelazowska M.A., McBride K.M., Kalluri R., Allison J., Petitprez F., Fridman W.H., Sautès-Fridman C., Hacohen N., Rezvani K., Sharma P., Tetzlaff M.T., Wang L, & Wargo J.A. (2020). B cells and tertiary lymphoid structures promote immunotherapy response. Nature, 577(7791), 549-555.

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Mass Cytometry Analysis of Immune Cells in Arthritis

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Murine Bone Marrow Cell Analysis

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Murine bone marrow was collected from both femurs and cells were immediately incubated with 10 uM iododeoxyuridine for 1 hour at 37°C. RBCs were lysed with 1 ml RBC lysis buffer for 5 min at 4°C and single-cell suspensions were prepared for analysis by time-of-flight mass cytometry. Cell numbers and viability were assessed using trypan blue and the remaining cells were transferred to a 96-well plate for staining with 5 uM cisplatin for 5 min at room temperature. Quenching in 5% FCS/5mM EDTA quenching buffer in PBS was performed before centrifugation at 300xg for 3 min at 4°C. Fc receptor blocking with metal conjugated anti-mouse CD16/32 antibody for 30 min at 4°C followed and quenched in quenching buffer before antibody staining for 30 min at 4°C. Cells were washed three times in quenching buffer, resuspended in 4% PFA and stored at 4°C. The following day, cells were washed once in PBS, permeabilized in methanol for 10 min at 4°C and washed three times before staining with intracellular antibodies for 45 min at room temperature. Samples were washed thrice in permeabilization buffer (Thermo Fisher), resuspended in 4% PFA and stored at 4°C until analysis using a Helios instrument (Fluidigm).

Kapellos T.S., Baßler K., Fujii W., Nalkurthi C., Schaar A.C., Bonaguro L., Pecht T., Galvao I., Agrawal S., Saglam A., Dudkin E., Frishberg A., de Domenico E., Horne A., Donovan C., Kim R.Y., Gallego-Ortega D., Gillett T.E., Ansari M., Schulte-Schrepping J., Offermann N., Antignano I., Sivri B., Lu W., Eapen M.S., van Uelft M., Osei-Sarpong C., van den Berge M., Donker H.C., Groen H.J., Sohal S.S., Klein J., Schreiber T., Feißt A., Yildirim A.Ö., Schiller H.B., Nawijn M.C., Becker M., Händler K., Beyer M., Capasso M., Ulas T., Hasenauer J., Pizarro C., Theis F.J., Hansbro P.M., Skowasch D, & Schultze J.L. (2023). Systemic alterations in neutrophils and their precursors in early-stage chronic obstructive pulmonary disease. Cell Reports, 42(6), 112525.

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Mass Cytometry Immunophenotyping of PBMCs

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Thawed PBMCs (∼3x10⁶ cells) were spun (300 g, 5 min) and resuspended in calcium magnesium-free phosphate buffered saline (PBS). 1μM Cisplatin (Fluidigm) was added for viability staining for 5 minutes before quenching the reaction with MaxPar Cell Staining Buffer (CSB, Fluidigm). After centrifugation (300 g, 5 min), cells were resuspended in CSB at a concentration of 60x10⁶ cells/mL and incubated (RT,10 min) with Fc receptor binding inhibitor before adding 26 MaxPar metal-conjugated antibodies (Fluidigm) against immunophenotypic markers for an additional 30-minute incubation at RT. Stained cells were then washed two times before resuspension in MaxPar fix and perm buffer with 125μM 191/193Ir intercalator for either an hour at RT or 4°C overnight. Cells were then washed twice with CSB and two times with Nuclease-Free water (Thermo Fisher Scientific) followed by filtering through 40μM strainers to remove aggregates. Cells were then counted and resuspended in Nuclease-Free water at ∼5x10⁵ cells/mL with 1:10 volume of four-element calibration beads (Fluidigm) and analyzed on a Helios instrument (Fluidigm) for 250,000 events for each donor at the NIEHS Flow Cytometry Center. Following the manufacturer’s instructions, downstream processing involved normalization by the calibration beads and fcs files were uploaded to Cytobank.

Martos S.N., Campbell M.R., Lozoya O.A., Wang X., Bennett B.D., Thompson I.J., Wan M., Pittman G.S, & Bell D.A. (2020). Single-Cell Analyses Identify Dysfunctional CD16+ CD8 T Cells in Smokers. Cell Reports Medicine, 1(4), 100054.

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Time-of-Flight Mass Cytometry of Immune Cells

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For time-of-flight mass cytometry, single cell suspensions of bone marrow, blood and spleen cells were stained with cell cycle marker iododeoxyuridine (IdU), viability marker cisplatin and surface and intracellular antibodies (online supplemental table S19). Expression levels of 38 markers were measured using a Helios instrument (Fluidigm), transformed with a logicle transformation 29 (link) and used for Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) using the naïve R implementation (umap V.0.2.7). Cells clusters were calculated using Rphenograph (Github JinmiaoChenLab V.0.99.1). 30 (link) Detailed methods are provided in the online supplemental file 1.
on August 31, 2024

Budden K.F., Shukla S.D., Bowerman K.L., Vaughan A., Gellatly S.L., Wood D.L.A., Lachner N., Idrees S., Rehman S.F., Faiz A., Patel V.K., Donovan C., Alemao C.A., Shen S., Amorim N., Majumder R., Vanka K.S., Mason J., Haw T.J., Tillet B., Fricker M., Keely S., Hansbro N., Belz G.T., Horvat J., Ashhurst T., van Vreden C., McGuire H., Fazekas de St Groth B., King N.J.C., Crossett B., Cordwell S.J., Bonaguro L., Schultze J.L., Hamilton-Williams E.E., Mann E., Forster S.C., Cooper M.A., Segal L.N., Chotirmall S.H., Collins P., Bowman R., Fong K.M., Yang I.A., Wark P.A.B., Dennis P.G., Hugenholtz P, & Hansbro P.M. (2024). Faecal microbial transfer and complex carbohydrates mediate protection against COPD. Gut, 73(5).

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Mass Cytometry Immunophenotyping of PBMCs

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Thawed PBMCs (~3×10⁶ cells) were spun (300 g, 5 min) and resuspended in calcium magnesium-free phosphate buffered saline (PBS). 1μM Cisplatin (Fluidigm) was added for viability staining for 5 minutes before quenching the reaction with MaxPar Cell Staining Buffer (CSB, Fluidigm). After centrifugation (300 g, 5 min), cells were resuspended in CSB at a concentration of 60×10⁶ cells/mL and incubated (RT,10 min) with Fc receptor binding inhibitor before adding 26 MaxPar metal-conjugated antibodies (Fluidigm) against immunophenotypic markers for an additional 30-minute incubation at RT. Stained cells were then washed two times before resuspension in MaxPar fix and perm buffer with 125μM 191/193Ir intercalator for either an hour at RT or 4°C overnight. Cells were then washed twice with CSB and two times with Nuclease-Free water (Thermo Fisher Scientific) followed by filtering through 40μM strainers to remove aggregates. Cells were then counted and resuspended in Nuclease-Free water at ~5×10⁵ cells/mL with 1:10 volume of four-element calibration beads (Fluidigm) and analyzed on a Helios instrument (Fluidigm) for 250,000 events for each donor at the NIEHS Flow Cytometry Center. Following the manufacturer’s instructions, downstream processing involved normalization by the calibration beads and fcs files were uploaded to Cytobank.

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