To examine different cell populations within human melanoma patient PBMC and mouse Pmel-1 splenocytes, cell surface flow cytometry was performed as previously described.27 (link) Human melanoma patient PBMC were stained with the following anti-human antibodies from BD: Fixable Viability Stain 575V, CD3 APC-R700, CD19 APC-H7, CD47 APC, PD-1 BV 421, and CD8 BUV496. Pmel-1 splenocytes were stained with dilutions of the following anti-mouse antibodies from BD: CD19 BV421, Fixable Viability Stain 575V, CD64 BV786, CD8a APC-H7, and CD3 BUV496. Flow cytometry was performed on the BD LSRFortessa X-20 Analyzer, and analysis was performed on FCS Express software (online supplemental figures 3C and 3D ).
Fixable viability stain 575v
Manufactured by BD
Sourced in United States
Fixable Viability Stain 575V is a fluorescent dye used for the identification and quantification of viable cells in flow cytometry applications. It binds to cellular proteins, allowing for the reliable detection of live and dead cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (2 mentions)
Lsrfortessa system, by BD (2 mentions)
Cd19 apc h7, by BD (1 mentions)
Cd8a apc h7, by BD (1 mentions)
Cd19 bv421, by BD (1 mentions)
Cd3 apc r700, by BD (1 mentions)
Dnase, by Merck Group (1 mentions)
Fixation permeabilization kit, by BD (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Liberase, by Merck Group (1 mentions)
Facs symphony flow cytometer, by BD (1 mentions)
Pharm lyse buffer, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Brefeldin a, by BD (1 mentions)
Cd28 ecd, by Beckman Coulter (1 mentions)
Cd3 alexa fluor 700, by Thermo Fisher Scientific (1 mentions)
Bc 5000, by Mindray (1 mentions)
Igd pe, by BioLegend (1 mentions)
8 protocols using fixable viability stain 575v
1
Multiparametric Flow Cytometry Analysis of Immune Cells
2
High-parameter flow cytometry analysis of immune cells
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High-parameter flow cytometry was performed on BAL cells and PBMCs at preinfection, at pretreatment, during treatment, at posttreatment, and at necropsy as previously described (17 (link), 34 (link), 66 (link), 74 (link), 77 (link)). The single cells prepared from lung, BAL, PBMCs, and other tissues were stained with surface and intracellular markers to study T cell phenotypes. Tissues obtained at necropsy were digested using Liberase and DNase (both Sigma-Aldrich), filtered, and subjected to RBC lysis (ACK Lysis Buffer, Gibco). The cells were then counted and used for staining for flow cytometry. The cells were first stained with extracellular/surface antibodies: CD3, CD4, CD8, CD45, CD28, and CD95 for 25 minutes at room temperature, followed by the Fixable Viability Stain 575V (BD Biosciences). The cells were then fixed and permeabilized using Fixation/Permeabilization Kit (BD Biosciences) for 30 minutes at 4°C. Subsequently, the cells were stained with intracellular antibody (Ki67) to study the T cell proliferation. Cells were then washed and acquired on a BD FACSSymphony flow cytometer. Analysis was performed using FlowJo (v10.5.3) using previously published gating strategies (Supplemental Figure 4 ) (30 (link), 32 (link)–34 (link)). The details of antibodies used in flow cytometry experiments are provided (Supplemental Table 3 ).
3
Flow Cytometry Analysis of Single-Cell Samples
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SCS were processed both with and without red blood cell lysis by BD Pharm Lyse buffer following manufacturer’s instructions prior to staining. Briefly, SCS and enriched cell populations were washed with PBS before staining with Fixable Viability Stain 575V (BD Biosciences) at 1:1,000 dilution for 15 min at RT. Then, cells were washed with PBS -1% FBS (Gibco) and incubated for 15 min at RT with antibodies against surface markers (Supplementary Data Sheet 2 Supplementary Data Sheet 3
4
Cytokine Responses in Cutaneous Leishmaniasis
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Plasma IFN-γ was measured using BioPlex-220 (Bio-Rad Laboratories) with Cytokine Grp-I-panel 27-plex. Peripheral blood mononuclear cells from a subset (n = 40) of untreated phase 2 CL patients were separated from heparinized blood over Ficoll and used to examine T-cell responses by IFNG-AS1 genotype. Cells (1 × 106 cells/mL) were stimulated: 37°C/5% CO2, 10 μg/mL L. braziliensis (strain MHOM/BR/2001) log-phase promastigote soluble Leishmania antigen, 1 μg/mL purified NA/LE anti-human CD28 (clone CD28.2, BD Biosciences, San Jose, California) for 15 hours, then brefeldin A (BD Biosciences) for 4 hours. Washed cells (phosphate-buffered saline/0.2% bovine serum albumin) were incubated (4°C; 30 minutes) with BUV661 anti-human CD3 monoclonal antibody (UCHT1 clone, BD Biosciences). Cells were fixed, washed, permeabilized using BD Cytofix/Cytoperm, and incubated (4°C; 30 minutes) with BV605 anti-human IFN-γ (B27 clone) and BUV395 anti-human TNF-α (MAB11 clone, BD Biosciences) in permeabilization buffer. Live/dead cells were distinguished using Fixable Viability Stain 575V (BD Biosciences). Data were acquired by BD FACSymphony A5 flow cytometry and analyzed using FlowJo 10.6.1 software (BD Biosciences), and differences between genotypes determined using nonparametric Kruskal-Wallis analysis of variance (ANOVA) with multiple comparisons.
5
Multiparametric Blood Cell Analysis
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Blood was collected from the inguinal veins at the indicated time points (Fig. 1A ) and hematological examination was conducted using an autohematology analyzer (Mindray BC-5000; Mindray, Shenzhen, China). Flow cytometric analysis was performed using a BD LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo v10.7.1 (BD Biosciences), as previously described (8 (link)). To exclude dead cells, blood was first stained with Fixable Viability Stain 575V (BD Biosciences) for 20 min at room temperature. For surface staining, cells were stained with the following antibodies for 30 min at 4°C: CD3 (Alexa Fluor 700; Invitrogen, Waltham, MA, USA), CD20 (APC/Cyanine7; Invitrogen), CD27 (PE/Cyanine7; Invitrogen), IgD (PE; BioLegend Inc., San Diego, CA, USA), IgM (FITC; SouthernBiotech, Birmingham, AL, USA), IgG (V450; BD Bioscience), CD4 (V500; Invitrogen), CD8 (V450; Invitrogen), CD95 (PE/Cyanine5; Invitrogen), and CD28 (ECD; Beckman Coulter, Brea, CA, USA). The cells were washed with permeabilization wash buffer and fixed with 1% paraformaldehyde. Data were acquired using an LSR Fortessa system (BD Biosciences) and analyzed using FlowJo v10.7.1 (BD Biosciences).
6
Multiparameter flow cytometry analysis
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Cells were stained with 10% mouse serum (Sigma‐Aldrich), 10% rat serum (Sigma‐Aldrich) and 10% MACS buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) at a 1:1:1 ratio for 10 min at 4°C to block the non‐specific binding, as described in a previous study.
38 (link) Cells were then washed with PBS and performed LiveDead staining with Fixable Viability Stain 575 V (BD Biosciences, San Jose, USA) or Zombie Aqua™ Fixable Viability Kit (Biolegend, San Deigo, USA) for 10 min at 4°C in the dark. Cells were subsequently washed with MACS buffer and stained with other cell surface antibodies for 15–20 min at 4°C. The expression of NK cell receptors and immune checkpoints on NK cells were analysed using a 21‐colour flow cytometry panel (Supplementary table1 ) and the expression of the corresponding ligands on the tumor cells was analysed using a 20‐colour flow cytometry panel (Supplementary tables 2 and 3 ). The cells were then washed and resuspended in MACS buffer and acquired on a FACSymphony A5 instrument (BD Biosciences).
38 (link) Cells were then washed with PBS and performed LiveDead staining with Fixable Viability Stain 575 V (BD Biosciences, San Jose, USA) or Zombie Aqua™ Fixable Viability Kit (Biolegend, San Deigo, USA) for 10 min at 4°C in the dark. Cells were subsequently washed with MACS buffer and stained with other cell surface antibodies for 15–20 min at 4°C. The expression of NK cell receptors and immune checkpoints on NK cells were analysed using a 21‐colour flow cytometry panel (Supplementary table
7
Flow cytometry of DENV-infected PBMCs
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Following infection with AF594-labelled DENVs, PBMCs of humans or non-human primate were stained first with Fixable Viability Stain 575V (BD Biosciences, USA) at 20 °C for live/dead cell gating, followed by surface staining with CD14, HLA-DR, CD16, CD20, and CD3. PBMCs were then analysed using a LSR Fortessa system (BD Bioscience) and FlowJo software v10.7.1.
8
Comprehensive Immune Cell Profiling
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Spleen, lymph nodes and/or tumors were harvested and passed through a 70 µm metal mesh and red blood cell lysed. Resulting single cell suspensions were stained with monoclonal antibodies specific for mouse CD8α (53-6.7), CD45.1 (A20), Vα2 (B20.1), IFNγ (XMG1.2), PD-1 (29F-1412), MHCI (AF6-88.5), CD45 (30-F11), and/or NK1.1 (PK1136) (all from BD Biosciences). For ex vivo cytokine production assays, splenocytes were first restimulated with 1 µM gB498-505 peptide for 1 h at 37 °C prior to addition of 0.22 mg Brefeldin A (BD GolgiPlug™, BD Biosciences) for a further 4 h. Following surface stain, cells were fixed with 4% paraformaldehyde prior to permeabilization with Permeabilization Buffer (eBioscience) and staining with IFNγ. Cells were stained with Fixable Viability Stain 575V at 1:20,000 (BD Biosciences) prior to surface staining or propidium iodide (PI; Sigma) immediately prior to acquisition to exclude dead cells. Cells were analyzed using the BD LSRFortessa and FlowJo software (BD Biosciences/TreeStar).
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