Compensated FCS3.0 files were exported from BD Facs Diva onto Flowjo Version 10. Manual gating was then performed using the gating strategies described in Supplementary Figures 2 , 3 . Automated analyses were performed using Flowjo Plugin Downsample version 3.0 to obtain similar events across several samples. After this, concatenations were carried out to obtain a single file. Later, the Flowjo Plugin Fast Fourier Transform-accelerated Interpolation-based t-SNE (FIt-SNE) was then executed on the resultant single concatenated FSC file at the following settings (t-SNE dimensions = 2, Nearest neighbors = Approximate, Perplexity = 20.0, and Maximum iterations = 3000) (Linderman et al., 2017 ).
Facsdiva
Manufactured by FlowJo
Sourced in United States
The FACSDiva is a flow cytometry system that allows for the analysis and sorting of cells. It is designed to provide high-performance data acquisition, analysis, and sorting capabilities for a wide range of applications in biological research and clinical settings.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (6 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Facsdiva, by BD (3 mentions)
Click it plus edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (2 mentions)
Fxcycle violet dna content stain, by Thermo Fisher Scientific (2 mentions)
Lsr dual fortessa flow cytometer, by BD (2 mentions)
Dnase 1, by New England Biolabs (2 mentions)
Anti ssea1 efluor 660, by Thermo Fisher Scientific (2 mentions)
Anti cd44 fitc im7, by Thermo Fisher Scientific (2 mentions)
Anti cd24 vioblue clone 32d12, by Miltenyi Biotec (2 mentions)
Percp cy5.5 conjugated streptavidin, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Facsaria 2, by BD (2 mentions)
Accumax, by STEMCELL (2 mentions)
Lsr 2, by BD (2 mentions)
Celltrace far red, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Alexa fluor 700 anti human cd3, by BioLegend (1 mentions)
20 protocols using facsdiva
1
Automated High-Dimensional Flow Cytometry Analysis
2
Cell Surface Staining and Flow Cytometry
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Cell staining was performed as previously described [32 (link)]. Briefly, surface staining was performed with the following antibody panel (Additional file 1 : Table S3) for 30 min at 4 °C. After washing with 1X phosphate buffered saline (PBS), cells were fixed and permeabilized using Cytofix/Cytoperm buffer set for 20 min at room temperature in the dark. Cells were washed twice with 1X FACS buffer and re-suspended in 200μL of FACS buffer. Fluorescence was measured using a BD FACSymphony cell analyzer with FACSDiva version 8.0.1 software (FLowJo LLC). Compensation, gating, and analysis were performed using FlowJo (versions 9 and 10). Reagents used for flow cytometry are listed in Additional file 1 : Table S3.
3
Microglia-CD8+ T Cell Interaction Assay
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After animal sacrifice at defined times p.i. and leukocytes enriched on a Percoll gradient as previously described (Blanc et al., 2014 (link)). Cell were sorted at the University of Utah Flow Cytometry Core based using the following markers, CD45lo, CD11b+, F4/80+. Microglia were then plated in a 96-well flat bottom plate coated with Poly-D-Lysine (Sigma Aldrich) and incubated with the S510 peptide overnight. Spleens were collected from MHV-DM infected SPF mice 7 D.P.I., viral-specific CD8 +T cells were sorted at the University of Utah flow cytometry core. T cells were stained using Far Red Cell Trace or CFSE (Thermo Fisher), CD8 +T cells were then co-cultured with microglia for 72 hr, samples were analyzed using BD LSRFortessa and FACSDiva software and data was measured using FlowJo.
4
Flow Cytometry Analysis of SARS-CoV-2 Infection
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Flow cytometry data were collected from the BD LSRFortessa using FACSDiva software and analyzed using FlowJo version 10. The n listed in the figure legends represents individual infections. Statistical analysis by multiple t-tests to compare differences in means of percent positive cells from 0 to 24 h, 0 to 48 h, and 24 to 48 h was performed using Graphpad Prism version 8. MeV N gene expression measured by rRT-PCR was normalized to RNaseP (RPPH1) and the 2−ΔCt calculated. Statistical analysis by multiple t-test to compare MeV N gene expression at 0 to 24 h, 0 to 48 h and 24 to 48 h was performed using Graphpad Prism version 8. Cytokine MFI was converted to pg/mL concentration using the included standard according to manufacturer’s recommendations. Increases in cytokine concentration were determined by subtracting detectable cytokine at 0-h timepoints. Statistical analyses for cytokine production were performed to determine differences between infection with EZ-GFP compared to infection with FL-15 or changes within each condition between 24- and 48-hours-post-infection. Differences were checked for significance using multiple t-tests.
5
Quantifying DNA Replication and Cell Cycle
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To measure DNA replication and cell cycle stage, EdU (5-ethynyl-2´-deoxyuridine) was added to cells at 10 nM for 1.5 h before harvesting. 1 confluent well of a 6-well plate of HCT116 cells were harvested and processed as per manufacturer’s instructions for the Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (Thermo Fisher cat: C10634 ). Per manufacturer’s instructions, FxCycle Violet DNA content stain (Thermo Fisher cat: F10347 ) was added after the Click-iT reaction at 1:1000 dilution before quantifying on a BD LSR Dual Fortessa flow cytometer. Alexa Fluor 647 was measured in the 670-30 Red C-A Channel and FxCycle Violet Stain was measured in the 450-50 Violet F-A Channel. Analysis was performed using FACS DIVA and FlowJo V10 (FlowJo, LLC) software.
6
Purification of CD4+CD25- T Cells
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CD4 + CD25‐T (mentioned as CD4+ T cells in the manuscript for convenience unless otherwise specified) cells were purified from the PBMCs using a CD4 + CD25+ T cell isolation kit based on a two‐step negative isolation protocol, as per the manufacturer's prescription (Stem cell technologies). The kit separates CD4 + CD25+ and CD4 + CD25‐ T cells using positive and negative selection respectively. We collected negatively selected cell population and assessed cellular purity by immunophenotyping using anti‐human‐CD3 Alexa Fluor 700 (Clone OKT3), CD4 APC‐Cy7 (clone RPA‐T4), CD25 PE‐Cy7 (clone BC96) (Biolegend Inc., San Diego, CA, USA), and FoxP3‐BD Horizon V450 (Clone 236A/E7) (BD) antibodies on a flow cytometer, (LSR™ Fortessa, BD Biosciences, San Jose, CA, USA). Purity of isolated cells (CD3 + CD4 + CD25‐FoxP3‐) ranged between 90‐98% (Figure S1 A‐C). All data were acquired on the same flow cytometer using FACS Diva™ software version 7.0 (BD Biosciences) and analysis was done by FACS Diva™ or Flowjo™ version 10.5.3 9.0 (Flowjo LLC, Ashland, OR, USA).
7
Flow Cytometry Analysis of SSEA1, CD44, and CD24
8
Quantifying Intracellular Hydrogen Peroxide
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The intracellular hydrogen peroxide levels were measured via staining with the oxidant-sensitive fluorescent probe CM-H2DCFDA (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester) (Molecular Probes, Eugene, OR, USA). The cells were incubated with OH-dDHL and then stained with CM-H2DCFDA for 30 min at 37 °C in the dark. The stained cells were immediately analyzed using the FACS Canto II with FACS Diva, and the results were analyzed using FlowJo software.
9
Analyzing Virus-Specific T Cell Responses
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After animal sacrifice and perfusion with ice cold PBS, half of the brain of mice was collected and homogenized at defined times p.i. and leukocytes enriched on a Percoll gradient as previously described (Blanc et al., 2014 (link)). Cells were stained using the following antibodies: CD4 (GK1.5, Biolegend), CD3 (145–2 C11, Biolegend), IFN-γ (XMG1.2, Biolegend), FoxP3 (FJK-16s, eBioscience), IL-10 (JES5-16E3, Biolegend), CD8 (53-6.7, Biolegend), CD45 (30-F11, Biolegend), F4/80 (BM8, Biolegend), CD11b (M1/70, Biolegend), CD40 (HM40-3, Biolegend), CD86 (GL-1, Biolegend), MHCI (M1/42, Biolegend), and MHCII (M5/114.15.2, Biolegend). Virus-specific T cells were determined via flow cytometric analysis through either intracellular staining for IFN-γ or defined tetramers specific for immunodominant CD4 +and CD8+T cell-specific viral epitopes (Chen et al., 2014 (link); Stiles et al., 2006 (link)). Samples were analyzed using BD LSRFortessa and FACSDiva software and data was measured using FlowJo.
10
Multiparametric Flow Cytometry Analysis
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For determination of various cell populations in the iFA model, the cells were dissociated using Accumax (Stem Cell Technologies) for 5 minutes, pelleted and resuspended in FACS buffer (3% Fetal Bovine Serum /PBS). 1 × 106 cells were incubated with the appropriate conjugated antibody for 20 minutes at 4°C under shaking conditions. The cells were washed with the FACS buffer and acquired using a flow cytometer (BD LSRII) and analyzed using FACS Diva and FlowJo softwares.
For dissociation of human lung slice cultures, 48 hours after DMSO or AA5 treatment, the samples were dissociated using the Multi Tissue Dissociation Kit (Milteny) (2.35 mL of DMEM, 100 μL of Enzyme D, 50 μL of Enzyme R, and 12.5 μL of Enzyme A) using gentleMACS Octo Dissociator at 37°C for 40 minutes. The cell suspension was passed though 40micron filter and pelleted. Erythrocytes were lysed using the Red Blood Lysis Solution (Milteny) for 2 minutes, pelleted and re-suspended in FACS buffer. Total cells were stained using the antibodies for 30 minutes followed by washing. The cells were resuspended in FACS buffer and analyzed by Flow Cytometry using the FACS Diva (BD Biosciences) and FlowJo softwares.
For dissociation of human lung slice cultures, 48 hours after DMSO or AA5 treatment, the samples were dissociated using the Multi Tissue Dissociation Kit (Milteny) (2.35 mL of DMEM, 100 μL of Enzyme D, 50 μL of Enzyme R, and 12.5 μL of Enzyme A) using gentleMACS Octo Dissociator at 37°C for 40 minutes. The cell suspension was passed though 40micron filter and pelleted. Erythrocytes were lysed using the Red Blood Lysis Solution (Milteny) for 2 minutes, pelleted and re-suspended in FACS buffer. Total cells were stained using the antibodies for 30 minutes followed by washing. The cells were resuspended in FACS buffer and analyzed by Flow Cytometry using the FACS Diva (BD Biosciences) and FlowJo softwares.
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