Lsrii hts, by BD - Product details

1

Single-cell RNA-seq Immune Cell Isolation

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Single-cell suspensions were added a live/dead cell staining with blue
reactive dye (1:250, L23105; Invitrogen (Thermo Fisher Scientific, Carlsbad,
USA) and Brilliant Violet 510 anti-CD45 antibody (1:100, 103137; Biolegend, San
Diego, USA) and isolated using the BD FACS Aria Iiu (BD Biosciences, San Jose,
USA). During cell sorting, cellular debris were excluded with FSC and SSC gating
and dead cells excluded with UVB channel negative selection. CD45⁺cells were then positively selected and purified and processed for single-cell
RNA sequencing as described in [25 (link)].
Flow-cytometric analysis was performed using the BD LSRII HTS (BD Biosciences,
San Jose, USA). In addition to the live/dead and CD45 staining, Brilliant Violet
785 anti-Fcgr4 (1:100, 149535; Biolegend, San Diego, USA) and PE/Dazzle 594
anti-CD31 (1:100, 102429; Biolegend, San Diego, USA) antibodies were used. Data
was analyzed using Flowjo version 10.4.2.

Weinstock A., Brown E.J., Garabedian M.L., Pena S., Sharma M., Lafaille J., Moore K.J, & Fisher E.A. (2019). Single-Cell RNA Sequencing of Visceral Adipose Tissue Leukocytes Reveals that Caloric Restriction Following Obesity Promotes the Accumulation of a Distinct Macrophage Population with Features of Phagocytic Cells. Immunometabolism, 1, e190008.

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2

Single-cell RNA-seq Immune Cell Isolation

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Single-cell suspensions were added a live/dead cell staining with blue
reactive dye (1:250, L23105; Invitrogen (Thermo Fisher Scientific, Carlsbad,
USA) and Brilliant Violet 510 anti-CD45 antibody (1:100, 103137; Biolegend, San
Diego, USA) and isolated using the BD FACS Aria Iiu (BD Biosciences, San Jose,
USA). During cell sorting, cellular debris were excluded with FSC and SSC gating
and dead cells excluded with UVB channel negative selection. CD45⁺cells were then positively selected and purified and processed for single-cell
RNA sequencing as described in [25 (link)].
Flow-cytometric analysis was performed using the BD LSRII HTS (BD Biosciences,
San Jose, USA). In addition to the live/dead and CD45 staining, Brilliant Violet
785 anti-Fcgr4 (1:100, 149535; Biolegend, San Diego, USA) and PE/Dazzle 594
anti-CD31 (1:100, 102429; Biolegend, San Diego, USA) antibodies were used. Data
was analyzed using Flowjo version 10.4.2.

Weinstock A., Brown E.J., Garabedian M.L., Pena S., Sharma M., Lafaille J., Moore K.J, & Fisher E.A. (2019). Single-Cell RNA Sequencing of Visceral Adipose Tissue Leukocytes Reveals that Caloric Restriction Following Obesity Promotes the Accumulation of a Distinct Macrophage Population with Features of Phagocytic Cells. Immunometabolism, 1, e190008.

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3

Flow Cytometric Cell Cycle Analysis

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Cells were prepared for propidium iodide staining as described but with modifications to optimize preparation of samples in 96 well plates. 30,000 cells were analyzed using the BD LSRII HTS. Flow-Jo Cell Cycle analysis was performed using the Dean-Jett-Fox model to estimate the mean G1 and G2 fluorescence peaks of each strain. Control parental 1N, 2N, 3N, and 4N strains were analyzed in triplicate with the evolved strains.

Selmecki A., Maruvka Y.E., Richmond P.A., Guillet M., Shoresh N., Sorenson A., De S., Kishony R., Michor F., Dowell R, & Pellman D. (2015). Polyploidy can drive rapid adaptation in yeast. Nature, 519(7543), 349-352.

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4

Annexin V-APC and Propidium Iodide Staining

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After induction of cell death, cells were stained with annexin V-APC (BioLegend, Cat# 640941, 1:50) and propidium iodide (Sigma, Cat# 4170,2 µg/mL) for 15 min at room temperature in annexin V binding buffer (BD). Flow cytometry analysis was performed using the FACS Calibur (BD) and LSR II HTS (BD). Data were analysed with FlowJo v.10 software.

Verboom L., Anderson C.J., Jans M., Petta I., Blancke G., Martens A., Sze M., Hochepied T., Ravichandran K.S., Vereecke L, & van Loo G. (2023). OTULIN protects the intestinal epithelium from apoptosis during inflammation and infection. Cell Death & Disease, 14(8), 534.

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5

Macrophage Efferocytosis Quantification

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RAW264.7 cells and human monocyte-derived macrophages were treated ± PCM for 16 hours. Media was aspirated and cells washed with PBS. Jurkat cells were irradiated with UV light for 15 minutes followed by incubation under normal culture conditions for 3 hours. During the third hour of incubation, cells were labeled with CypHer5E NHS ester (GE Life Sciences, Pittsburgh, PA). Cells were washed twice with PBS, suspended in RPMI + containing 0.2% fatty acid-free bovine serum albumin (Sigma-Aldrich, St. Louis, MO) and applied to macrophages at 5:1 Jurkat:macrophage for 40 minutes. Media was aspirated and cells were washed with ice-cold PBS. Macrophages were labeled with FITC anti-CD11b antibody (Biolegend, San Diego, CA) for 45 minutes, washed and analyzed with a LSRII HTS (BD Bioscience, Franklin Lakes, NJ) flow cytometer. Macrophages were first selected with CD11b, from which the CypHer5 positive events (efferocytotic events) were selected. Data analysis was performed using Flow Jo version 10.4.2.

Heffron S.P., Weinstock A., Scolaro B., Chen S., Sansbury B.E., Marecki G., Rolling C.C., El Bannoudi H., Barrett T., Canary J.W., Spite M., Berger J.S, & Fisher E.A. (2020). Platelet Conditioned Media Induces an Anti-inflammatory Macrophage Phenotype through EP4. Journal of thrombosis and haemostasis : JTH, 19(2), 562-573.

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6

Cell Cycle Analysis using Flow Cytometry

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For single-parameter DNA content analysis, Hoechst 33342 was added to healthy KBM7 cell culture suspension (<10⁶ cells/mL) for a final concentration of 10 μg/mL and cells were incubated in a 37 °C water bath for 20 min before flow cytometry analysis on a BD LSRII-HTS instrument. Acquired DNA content signals were analyzed in the FlowJo software (v9) for the proportion of cells in G1, S, and G2/M phases.
For double-parameter cell cycle analysis, cells were pulse labeled with 10 μM 5-bromo-2’-deoxyuridine (BrdU) for 2 h, fixed using the BD Pharmingen BrdU Flow Kit (Cat. No. 559619), treated with DNase to expose BrdU epitope, stained with an FITC-conjugated anti-BrdU antibody as well as 7-AAD for total DNA content, followed by flow cytometry analysis.

Liu-Lupo Y., Ham J.D., Jeewajee S.K., Nguyen L., Delorey T., Ramos A., Weinstock D.M., Regev A, & Hemann M.T. (2023). Integrated multi-omics analyses reveal homology-directed repair pathway as a unique dependency in near-haploid leukemia. Blood Cancer Journal, 13(1), 92.

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7

Platelet activation detection by flow cytometry

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WB was centrifuged (15 min, 210 × g) to produce platelet rich plasma. PLT activation was detected using a flow cytometer (LSRII‐HTS, BD Biosciences, Breda, the Netherlands)) after staining of PLTs with fluorescent CD62P‐FITC (P‐selectin, Beckman Coulter, Immunotech) as described before.²²

Osemwengie D., Lagerberg J.W., Vlaar R., Gouwerok E., Go M., Nierich A.P, & de Korte D. (2021). Recovery of platelet‐rich red blood cells and acquisition of convalescent plasma with a novel gravity‐driven blood separation device. Transfusion Medicine (Oxford, England), 32(1), 53-63.

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8

Flow Cytometry Analysis of Immune Cells

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Staining for flow cytometry analysis was performed by incubating single-cell suspensions with primary fluorochrome-labeled antibodies on ice for 30 min. Zombie Yellow Fixable Viability Kit (BioLegend 423103) was used to assess live/dead populations. Cells were fixed in 3.7% formaldehyde diluted in FACs buffer (PBS + 5% FBS) for 30 min at room temperature prior to analysis. For iNOS and ARG1 staining, cells were permeabilized and fixed with FoxP3/Transcription Factor Staining Buffer Set (eBioscience CNOO-5523) for 30 min at room temperature and stained overnight at 4 °C. All samples were washed once with FACs buffer prior to analysis on an LSRII HTS flow cytometer (BD Biosciences). Collected data were analyzed using FlowJo data analysis software (v10).

LaRue M.M., Parker S., Puccini J., Cammer M., Kimmelman A.C, & Bar-Sagi D. (2022). Metabolic reprogramming of tumor-associated macrophages by collagen turnover promotes fibrosis in pancreatic cancer. Proceedings of the National Academy of Sciences of the United States of America, 119(16), e2119168119.

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9

Flow Cytometry of HUVEC or FOCUS Cells

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For flow cytometry, HUVECs or FOCUS cells were trypsinized for ≥5 min, trypsin was quenched with medium, and the cells were run on a FACScan, FACSCalibur, LSRII HTS, or LSR-Fortessa HTS (BD) flow cytometer with or without the membrane exclusion dye propidium iodide for a set amount of time at a constant flow rate to determine cell number. FlowJo was used for post-acquisition analysis. DCFDA ROS detection kit (Abcam) was used according to the manufacturer's instructions.

Bent E.H., Gilbert L.A, & Hemann M.T. (2016). A senescence secretory switch mediated by PI3K/AKT/mTOR activation controls chemoprotective endothelial secretory responses. Genes & Development, 30(16), 1811-1821.

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10

Flow Cytometric Cell Cycle Analysis

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Cells were prepared for propidium iodide staining as described but with modifications to optimize preparation of samples in 96 well plates. 30,000 cells were analyzed using the BD LSRII HTS. Flow-Jo Cell Cycle analysis was performed using the Dean-Jett-Fox model to estimate the mean G1 and G2 fluorescence peaks of each strain. Control parental 1N, 2N, 3N, and 4N strains were analyzed in triplicate with the evolved strains.

Selmecki A., Maruvka Y.E., Richmond P.A., Guillet M., Shoresh N., Sorenson A., De S., Kishony R., Michor F., Dowell R, & Pellman D. (2015). Polyploidy can drive rapid adaptation in yeast. Nature, 519(7543), 349-352.

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Lsrii hts

Lab products found in correlation

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10 protocols using lsrii hts

Single-cell RNA-seq Immune Cell Isolation

Single-cell RNA-seq Immune Cell Isolation

Flow Cytometric Cell Cycle Analysis

Annexin V-APC and Propidium Iodide Staining

Macrophage Efferocytosis Quantification

Cell Cycle Analysis using Flow Cytometry

Platelet activation detection by flow cytometry

Flow Cytometry Analysis of Immune Cells

Flow Cytometry of HUVEC or FOCUS Cells

Flow Cytometric Cell Cycle Analysis

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