Helios 6.5.358 acquisition software

Manufactured by Standard BioTools

The Helios 6.5.358 acquisition software is a tool designed to control and manage the data acquisition process for laboratory equipment. It provides the core functionality to configure, initiate, and monitor the data collection from the hardware. The software is intended to serve as an interface between the user and the equipment, facilitating the data acquisition workflow.

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Lab products found in correlation

Iridium nucleic acid intercalator, by Standard BioTools (3 mentions) Cell strainer cap, by BD (2 mentions) Eq four element calibration beads, by Standard BioTools (2 mentions) Palladium metal barcoding reagents, by Standard BioTools (2 mentions) Helios mass cytometer, by Standard BioTools (2 mentions) 194pt monoisotopic cisplatin, by Standard BioTools (2 mentions) Debarcoder software, by Standard BioTools (1 mentions) Helios instrument, by Standard BioTools (1 mentions) Cell strainer cap tubes, by BD (1 mentions) Foxp3 fix and permeabilization kit, by Thermo Fisher Scientific (1 mentions)

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Helios software (4 citations)

6 protocols using helios 6.5.358 acquisition software

Mass Cytometry Data Analysis Pipeline

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Mass cytometry data files were concatenated and normalized to EQ4 bead signal using the Helios 6.5.358 acquisition software (Fluidigm). Normalized data were then debarcoded to deconvolve individual samples using the Helios 6.5.358 acquisition software or stand-alone Debarcoder software (Fluidigm). Data files associated with individual samples were then utilized for downstream analyses. Data were manually gated Live CD45⁺ Lin⁻ CD3ε⁺ T cells using FlowJo (TreeStar). Clustering was applied to further remove any non CD4⁺ or CD8⁺ T cells for input into archetypal analysis. See Tables S1 and S2 for analyses performed on each respective mass cytometry dataset. Mass cytometry datasets will be made publicly available through FlowRepository (https://flowrepository.org).

Wei S.C., Sharma R., Anang N.A., Levine J.H., Zhao Y., Mancuso J.J., Setty M., Sharma P., Wang J., Pe’er D, & Allison J.P. (2019). Negative costimulation constrains T cell differentiation by imposing boundaries on possible cell states. Immunity, 50(4), 1084-1098.e10.

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Comprehensive Mass Cytometry Protocol for B Cell Analysis

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The next day, samples were washed twice with cell staining buffer, re-suspended in 1 ml of MilliQ ddH2O, filtered through a 35-µm nylon mesh (5ml tubes with cell strainer caps, BD) and counted. Before analysis, samples were resuspended in MilliQ ddH2O supplemented with EQ four element calibration beads (Fluidigm®) at a concentration of 0.5-1.0 x 10⁶ cells/ml. Samples were acquired at 300 events per second on a Helios instrument (Fluidigm®) using the Helios 6.5.358 acquisition software (Fluidigm®). We collected a minimum range of 750,000 – 1.2 million cells per samples in order to maximise chances of detecting rarer B cell subsets. IL10 detection albeit low in the unstimulated mass cytometry cohort, was reflective of likely Breg populations given the surface phenotype. We performed corroborative work on a stimulated cohort of melanoma cells which showed no difference in IL10 when compared to the parallel unstimulated cohort (19 (link)). Individual.fcs files collected from each set of samples were concatenated using the. fcs concatenation tool from Fluidigm® (CyTOF normalisation software 2), and data were normalized based on EQ four element signal shift over time using the same tool.

Patel A.J., Khan N., Richter A., Naidu B., Drayson M.T, & Middleton G.W. (2023). Deep immune B and plasma cell repertoire in non-small cell lung cancer. Frontiers in Immunology, 14, 1198665.

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Comprehensive Immunophenotyping by Mass Cytometry

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2×10⁶ or fewer cells were incubated with 2% of FBS in PBS with 25 μg/mL of 2.4G2 antibody at 4°C for 10 min prior to surface staining with an antibody cocktail at 4°C for 30 min in a 50 μL volume. Cells were incubated with 2.5 μM 194Pt monoisotopic cisplatin (Fluidigm) at 4°C for 1 min. Cells were then washed twice with FACS buffer and barcoded using palladium metal barcoding reagents according to manufacturer’s protocol (Fluidigm). Subsequently, fixation and permeabilization were performed using the Foxp3 fix and permeabilization kit according to the manufacturer’s protocol (Fluidigm). Cells were then stained with an intracellular stain antibody cocktail (Foxp3, Ki67, granzyme B, T-bet, iNos, Eomes) for 30 min at room temperature. Cells were then washed twice with Foxp3 permeabilization buffer, twice with FACS buffer, and incubated overnight in 1.6% PFA PBS with 100 nM iridium nucleic acid intercalator (Fluidigm). Cells were then washed twice with PBS with 0.5% BSA, filtered, and washed twice with water with 0.1% BSA prior to analysis. Samples were analyzed using a Helios mass cytometer based on the Helios 6.5.358 acquisition software (Fluidigm).

Wang Y., Liu S., Yang Z., Algazi A.P., Lomeli S.H., Wang Y., Othus M., Hong A., Wang X., Randolph C.E., Jones A.M., Bosenberg M.W., Byrum S.D., Tackett A.J., Lopez H., Yates C.C., Solit D.B., Ribas A., Piva M., Moriceau G, & Lo R.S. (2021). Anti-PD-1/L1 lead-In before MAPK inhibitor combination maximizes antitumor immunity and efficacy. Cancer cell, 39(10), 1375-1387.e6.

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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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Multiparametric Immune Profiling by CyTOF

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2x10⁶ or fewer cells were incubated with 2% of FBS in PBS with 25 μg/mL of 2.4G2 antibody at 4°C for 10 min prior to surface staining with an antibody cocktail at 4°C for 30 min in a 50 μL volume. Cells were incubated with 2.5 μM 194Pt monoisotopic cisplatin (Fluidigm) at 4°C for 1 min. Cells were then washed twice with FACS buffer and barcoded using palladium metal barcoding reagents according to manufacturer’s protocol (Fluidigm). Subsequently, fixation and permeabilization were performed using the Foxp3 fix and permeabilization kit according to the manufacturer’s protocol (eBioscience). Cells were then stained with an intracellular stain antibody cocktail (Foxp3, Ki67, granzyme B, T-bet, iNos, Eomes) for 30 min at room temperature. Cells were then washed twice with Foxp3 permeabilization buffer, twice with FACS buffer, and incubated overnight in 1.6% PFA PBS with 100 nM iridium nucleic acid intercalator (Fluidigm). Cells were then washed twice with PBS with 0.5% BSA, filtered, and washed twice with water with 0.1% BSA prior to analysis. Samples were analyzed using a Helios mass cytometer based on the Helios 6.5.358 acquisition software (Fluidigm).

Hong A., Piva M., Liu S., Hugo W., Lomeli S.H., Zoete V., Randolph C.E., Yang Z., Wang Y., Lee J.J., Lo S.J., Sun L., Vega-Crespo A., Garcia A.J., Shackelford D.B., Dubinett S.M., Scumpia P.O., Byrum S.D., Tackett A.J., Donahue T.R., Michielin O., Holmen S.L., Ribas A., Moriceau G, & Lo R.S. (2020). Durable Suppression of Acquired MEK Inhibitor Resistance in Cancer by Sequestering MEK from ERK and Promoting Anti-Tumor T-cell Immunity. Cancer discovery, 11(3), 714-735.

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Mass Cytometry Profiling of B Cell Subsets

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The next day, samples were washed twice with cell staining buffer, resuspended in 1 ml of MilliQ ddH₂O, filtered through a 35-µm nylon mesh (5-ml tubes with cell strainer caps, BD) and counted. Before analysis, samples were resuspended in MilliQ ddH₂O supplemented with EQ four-element calibration beads (Fluidigm^®) at a concentration of 0.5–1.0 × 10⁶ cells/ml. Samples were acquired at 300 events per second on a Helios instrument (Fluidigm^®) using the Helios 6.5.358 acquisition software (Fluidigm^®). We collected a minimum range of 750,000–1.2 million cells per sample in order to maximise the chances of detecting rarer B-cell subsets. IL-10 detection albeit low in the unstimulated mass cytometry cohort, was reflective of likely Breg populations given the surface phenotype. We performed corroborative work on a stimulated cohort of melanoma cells which showed no difference in IL-10 when compared to the parallel unstimulated cohort^{33 (link)}. Samples were acquired using two separate B-cell panels (Deep B-cell phenotyping and B-regulatory cell/T follicular helper cell panels). Individual.fcs files collected from each set of samples were concatenated using the.fcs concatenation tool from Fluidigm^® (CyTOF normalisation software 2), and data were normalised based on EQ four-element signal shift over time using the same tool.

Patel A.J., Willsmore Z.N., Khan N., Richter A., Naidu B., Drayson M.T., Papa S., Cope A., Karagiannis S.N., Perucha E, & Middleton G.W. (2022). Regulatory B cell repertoire defects predispose lung cancer patients to immune-related toxicity following checkpoint blockade. Nature Communications, 13, 3148.

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