Cells were washed in PBS and stained for viability using FVS510 (BD Biosciences) which was diluted 1:1,000 in PBS and 100 µL was added to each sample of cells and left in the dark at room temperature for 15 min and then washed twice in FACs buffer (PBS containing 0.5% BSA, 5 µM EDTA and 0.5% sodium azide). To reduce nonspecific binding, samples were incubated with 50 µL of polyclonal rat IgG (Sigma) (diluted 1:50 in FACs buffer) for 15 min on ice and protected from light, then washed once in FACs buffer by spinning at 400 g for 5 min and removing supernatant. Samples were then stained for flow cytometry by adding 50 µL of anti-CD44-FITC (clone IM7 at 1/200; Biolegend) in Brilliant Staining Buffer (BD Biosciences). Staining with TGMs used Alexa fluor-labeled proteins made as described above. TGMs labeled with AF488 and AF594 were used at a final concentration of 5 μg/mL. Samples that were also stained for intracellular antigens were fixed and permeabilized using Foxp3 Transcription Factor Buffer kit (Invitrogen, USA) and stained with anti-Foxp3-ef450 (clone FJK-16s, eBioscience, San Diego, California, USA, 1/100) in perm/wash buffer. Samples were then washed prior to analysis on a BD Celesta flow cytometer (BD Biosciences).
Fvs510
Manufactured by BD
Sourced in United States, Belgium
The FVS510 is a laboratory equipment designed for vacuum filtration applications. It features a stainless steel manifold and can accommodate up to 6 filter funnels simultaneously. The device provides a stable and consistent vacuum for efficient separation and collection of samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 fitc, by BD (2 mentions)
Cd3 apc h7, by BD (2 mentions)
Ebioscience fixation permeabilization buffers, by Thermo Fisher Scientific (2 mentions)
Ebioscience permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Cd4 percp, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Anti cd44 fitc clone im7, by BioLegend (1 mentions)
Brilliant staining buffer, by BD (1 mentions)
Celesta flow cytometer, by BD (1 mentions)
Polyclonal rat igg, by Merck Group (1 mentions)
Anti cd11b apc, by BD (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Anti f4 80 bv421, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Human fc block, by BD (1 mentions)
Fixation and permeabilization solution, by BD (1 mentions)
Stain buffer, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
17 protocols using fvs510
1
Multicolor Flow Cytometry Staining Protocol
2
Isolation and Characterization of Lung Immune Cells
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The whole lung was cut into pieces with ophthalmic scissors. The tissue was incubated with 0.1% type I collagenase (Sigma, MO, USA) at 37°C for 60 min. Then, the cells were homogenized through a 70 μm cell strainer (BD Biosciences). Isolated cells from lung tissue of mice were first incubated with FVS510 (BD Biosciences, CA, USA) for 15 min. After washing two times with 2% FBS, cells were incubated with monoclonal antibody against CD16/CD32 (BD Pharmingen, CA, USA) for 10 min to block Fc receptors. Next, cells were stained with BB515-anti-CD45 (BD Biosciences), BV421-anti-F4/80 (BD Biosciences), BV711-anti-Ly6G (BD Biosciences), APC-anti-CD11b (BD Biosciences), and PerCP-Cy5.5-anti-MHC-II (BD Biosciences) at 4°C for 20 min. The cells were fixed and permeabilized with 200 μl Perm/Wash Buffer (BD Biosciences). Intracellular staining of BV605-anti-CD206 antibody (BD Biosciences) was incubated at 4°C for 40 min. Specific cell types were identified as follows: neutrophil (CD45+CD11b+Ly6G+), macrophage (CD45+F4/80+), M1 macrophage (CD45+F4/80+MHC-II+), M2 macrophage (CD45+F4/80+CD206+). The data were analysed with Flow Jo software (version10.3, Tree Star, Inc., Ashland OR, USA).
3
Gastric Cancer Tissue Single-Cell Immune Profiling
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Freshly resected gastric cancer tissues were collected, digested and incubated with GolgiStop (BD Biosciences) at 37°C for 2 h. Then, the single‐cell suspensions were lysed with lysing buffer (BD Biosciences), protected from light, at room temperature for 15 min. The cells were stained with FVS510 (BD Biosciences) to determine live/dead cells. Afterwards, human Fc Block (BD Biosciences) was applied to block Fc receptor. Cells were stained for surface markers at 4°C for 30 min and protected from light. If necessary, cells were fixed and permeabilized with Fixation and Permeabilization Solution (BD Biosciences) at 4°C for 20 min. Intracellular cytokine staining was performed at 4°C for 30 min and protected from light. Stained cells were ultimately washed and resuspended in stain buffer (BD Biosciences). Flow cytometry (FC) or intracellular flow cytometry (ICFC) was performed by FACSCelesta or FACSAria III (BD Biosciences). Data were analysed by FlowJo (Tree Star).
4
Tumor and Immune Cell Profiling by Flow Cytometry
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Samples were prepared for flow cytometry as described previously54 (link). Briefly, single cell suspensions were generated from tumours using the mouse Tumour Dissociation Kit (Miltenyi) and splenocytes by mechanical dissociation. Cells were stained for viability (FVS510, BD Biosciences) and incubated with an Fc-blocking antibody (CD16/CD32, clone 2.4G2, BD Biosciences) prior to extracellular staining. Extracellular staining was carried out at room temperature (RT) for 20 min, samples were fixed in 1% paraformaldehyde (PFA) and stored overnight at 4 °C until samples were acquired. For intracellular staining of IFN-γ, the Foxp3/Transcription Factor Staining Buffer Set (eBioscience™) was used following the manufacturer’s instructions. For YAC-1/NK co-culture experiments, cells were stained with Ethidium Homodimer (EthD-1, Invitrogen) to differentiate live/dead cells prior to flow cytometric analysis and were acquired on the same day. Samples were acquired on a BD LSR Fortessa or BD FACS-Celesta flow cytometer. Data were analyzed using FlowJo software (v10.8.1).
5
Multiparametric Flow Cytometry of Immune Cells
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Single cell suspensions of lungs, spleen, and LNs from immunized mice were washed with PBS and resuspended in FACS-buffer [PBS supplemented with 1% (v/v) fetal calf serum and 0.1% (w/v) sodium azide]. Following treatment with Fc-block (BD Biosciences, Lyngby, Denmark), the cells were stained for 30 min at 4°C for surface markers using mAbs (Supplementary Table S1 ). Dead cells were excluded by using the fixable viability dyes FVS510 or FVS700 (BD). The cells were washed twice, resuspended in FACS buffer and analyzed using an LSRFortessa flow cytometer (BD). Gates for the surface markers are based on fluorescence-minus-one controls. The gating strategy used for identifying distinct cell populations in the lungs, the spleen, and the draining lymph nodes is based on previous reports (26 (link)–28 (link)) and is further described in the Supplementary Figures S1 –S3 . All flow cytometry analyses were performed using the FlowJo software v10 (Tree Star, Ashland, OR, United States).
6
Quantifying TβRIII Expression in Cells
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To analyze surface TβRIII expression on cancer cells and PBMCs, aliquots of 1 × 106 cells were incubated with fixable viability stain 510 (FVS510, BD Biosciences, Erembodegem, Belgium) to discriminate viable from non-viable cells for 15 min, followed by centrifugation and two washes with PBS. Cells were then stained with PE anti-human TβRIII polyclonal antibody (LSBio, Huissen, The Netherlands). Flow cytometric analysis was performed on a BD FACSCanto II flow cytometer. Data were analyzed using the FlowJo software (Tree Star).
7
Comprehensive Immune Cell Phenotyping by Flow Cytometry
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Single-cell suspensions were prepared from the thymus, spleen, and lymph nodes. Fluorochrome-conjugated antibodies against CD4 (RM4-5; eBioscience), CD8 (53-6.7; BD Biosciences), CD3 (17A2; BioLegend), CD44 (IM7; BD Biosciences), CD62L (MEL-14; BioLegend), B220 (RA3-6B2; BioLegend), NK1.1 (PK136; BD Biosciences), CD122 (TM-β1; BD Biosciences), CD69 (H1.2F3; eBioscience), TCRβ (H57-597; eBioscience), and CD24 (M1/69; eBioscience) were used for staining. FVS510 (BD Biosciences) was used for dead/live cell staining. For intracellular staining, cells were first stained with surface antibodies and then fixed and permeabilized with freshly prepared fixation/permeabilization working solution (BD Biosciences) according to the manufacturer’s instructions. Then, the cells were stained with anti-IFNγ (XMG1.2; eBioscience), anti-H3K4me1 (D1A9; CST) or anti-H3K4me2 (Y47; Abcam) diluted in permeabilization buffer. Data were acquired on an LSRFortessa (BD Biosciences) and analyzed with FlowJo Software (version 10.6.2). For cell sorting, single-cell suspensions isolated from the thymus were sorted on a FACSAria II (BD Biosciences).
8
Multiparameter Flow Cytometry of Immune Cells
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Following perfusion with PBS, the spleen, CSF (5 μl per mouse) and spinal cord were isolated from PBS or CRE-TAT treated mice at day 14 post-immunization. Cell suspensions of the spleen and spinal cord were prepared and incubated with ACK buffer (C3702, Beyotime) to lyse red blood cells. With a centrifugation at 1500 rpm for 5 min, the cells were resuspended with 100 μl of staining buffer (70-S1001, Multisciences). Next, 10 μl of Clear Back (FcR blocking, MTG-001, MBL) were mixed with 50 μl of cell suspension (≤ 1 × 106) and incubated for 5 min at room temperature. Then, the samples were stained with fluorochrome-conjugated monoclonal antibodies against FVS510 (564406, BD Pharmingen), CD3-FITC (11-0032-82, Invitrogen), CD4-PerCP-Cy5.5 (550954, BD Pharmingen) and CD196-AF647 (561753, BD Pharmingen) for 30 min at 4 °C. Next, the staining buffer (3 ml per sample) was added into the sample solution and mixed well. Following a centrifugation at 1500 rpm for 5 min, the cells were resuspended with 200 μl of staining buffer. At last, the stained cells were acquired with a flow cytometer (NovoCyte Quanteon, Agilent) and analyzed with NovoExpress software (Agilent).
9
Flow Cytometric Analysis of Vaccine-Induced Immune Responses
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Flow cytometry was used to evaluate the frequency of production of IFN‐γ, TNF‐α, and IL‐2 by CD4+ or CD8+ cells in the vaccine and control groups. Splenocytes were isolated and seeded at 106 cells/well in duplicate in serum‐free medium and stimulated for 8 h with 50 μg/ml of SA14‐14‐2 or YFV‐17D NS3 peptide antigen or with 100 μg/ml of total Vero cellular antigen at 37°C and 5% CO2. Also, 5 μg brefeldin A (BFA) (BioLegend, Cat# 420601) was added to block IFN‐γ, TNF‐α, and IL‐2 secretions at 6 h before the endpoint. Afterward, cells were harvested in a tube and stained with PE‐conjugated anti‐mouse CD3e, FITC‐conjugated anti‐mouse CD4, and PerCP/Cy5.5‐conjugated anti‐mouse CD8a antibodies (BioLegend, Cat# 100206, 100510, 100734), followed by 20‐min incubation in the dark. Intracellular cytokines were detected after 30‐min incubation with APC‐Cy7‐conjugated rat anti‐mouse IL‐2 (BD, Cat# 560547), APC‐conjugated anti‐mouse IFN‐γ (BioLegend, Cat# 505809), and PE/Cyanine7‐conjugated anti‐mouse TNF‐α (BioLegend, Cat# 506324) antibodies in the dark. FVS510 (BD, Cat# 564406) was used to distinguish viable cells. At least 50,000 events were analyzed with a BD LSRII flow cytometer and Summit software.
10
Analyzing T Cell Phenotypes by ICS
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To evaluate the phenotypes of T cells that responded to stimulation in IFN-γ ELISPOT assays, we conducted intracellular cytokine staining (ICS). The in vitro stimulation was similar to that described above in the IFN-γ ELLISPOT section. Briefly, after 12-14 days in vitro culture, PBMCs were restimulated with peptide. After 1 h, Brefeldin A was added to inhibit protein transport. After 4 additional hours of incubation, the cells were stained with T-cell surface markers CD4-PerCP (cat. 345770), CD8- FITC (cat. 345772), CD3-APC-H7 (cat. 560275) and a dead cell marker FVS510 (564406) (all from BD Biosciences). Samples were then fixed and permeabilized using eBioscience™ Fixation/Permeabilization buffers (eBioscience, cat. 00-5123-43, 00-5223-56) and stained with IFNg-APC (cat.341117, BD Biosciences), TNFa-BV421 (cat.562783, BD Biosciences) in eBioscience permeabilization buffer (eBioscience, cat. 00-8333-56) and analyzed on FACSCanto™ II (BD Biosciences) using BD FACSDiva software version 8.0.2 as described previously (32 (link)).
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