Flow cytometry was performed to immunophenotype inflammatory cells entering the CNS using established protocols (3 (link), 13 (link)). In brief, single cell suspensions were generated from tissue samples by grinding with frosted microscope slides. Immune cells were enriched by a 2-step Percoll cushion (90% and 63%) and cells were collected at the interface of the two Percoll layers. Before staining with the fluorescent antibodies, isolated cells were incubated with anti-CD16/32 Fc block (BD Biosciences, San Jose, CA) at a 1:200 dilution. Immunophenotyping was performed using either Rat anti-mouse IgG or Armenian hamster anti-mouse IgG antibodies for the following cell surface markers: F4/80 (Serotec, Raleigh, NC), MHV S510-tetramer (NIH), MHV M133-Tetramer (NIH) and CD4, CD8, Ly6g, CD11b, IFN-γ (BD Biosciences, San Jose, CA) and Ly6c (eBioscience, San Diego, CA). The following flow cytometric gating strategies for employed for immunophenotyping inflammatory infiltrates in the brain: neutrophils (CD45hiCD11b+ Ly6G+); monocytes (CD45hiCD11b+Ly6C+Ly6G−, macrophages (CD45hiCD11b+F4/80+); microglia (CD45lo, CD11b+, F4/80lo); M133-specific CD4+ T cells (CD45hi, CD4+, M133-tetramer+); S510-specific CD8+ T cells (CD45hi, CD8+, S510-tetramer+).
Fc block anti cd16 32
Manufactured by BD
Sourced in United States, United Kingdom
The Fc block (anti-CD16/32) is a laboratory reagent that binds to the Fc receptors CD16 and CD32 on cells. This binding can be used to block these receptors, preventing potential interference in downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BD (5 mentions)
Cd19 fitc, by BD (4 mentions)
Facscanto flow cytometer, by BD (4 mentions)
Cd19 pe, by BD (3 mentions)
Percp cy5.5 cd4, by BD (3 mentions)
Cd4 pe, by BD (3 mentions)
Cd19 pe cy7, by BD (3 mentions)
Cd45 apc cy7, by BD (3 mentions)
Cd138 apc, by BD (3 mentions)
Fitc mhc 2, by BD (3 mentions)
Cd3 apc cy7, by BD (3 mentions)
Cd4 fitc, by BD (3 mentions)
Lsrfortessa, by BD (3 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Armenian hamster anti mouse igg for cd80 percp cy5.5 16 10a1, by BD (2 mentions)
Cd11c fitc n418, by BioLegend (2 mentions)
Cd4 fitc rm4 5, by BD (2 mentions)
Cd11b pe, by BD (2 mentions)
Percp cy5.5 cd45, by BD (2 mentions)
Percp cy5.5 mhcii, by BD (2 mentions)
33 protocols using fc block anti cd16 32
1
Flow Cytometry Analysis of Inflammatory CNS Infiltrates
2
Flow Cytometric Characterization of T-Cells and DCs
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Identification of surface molecule expression by flow cytometric analysis was conducted as previously described [29 (link)]. Briefly, samples were blocked by incubating purified anti-CD16/32 (Fc block, BD Biosciences) on ice for 15 minutes. Then, samples were stained with mouse specific T-cell markers or DC markers antibody cocktails in staining buffer (1% BSA in 1xPBS) on ice for 45–60mins. T-cell marker cocktail: CD4-FITC, CD8-PE, CD28-PECy5, CD-25-PECy7, and CTLA-4-APC. DC marker cocktail: H2Kb-FITC, I-Ab-PE, CD86-PECy5, CD11c-PECy7, and CD80-APC. Stained samples were fixed with 4% paraformaldehyde on ice for 15 mins. Stained and fixed samples were stored in 500μL of staining buffer. Samples were read on Gallios Flow Cytometer (Beckman-Coulter). Analysis was conducted with Cyflogic.
3
Flow Cytometry of Immune Cell Subsets
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Cells were resuspended in staining buffer (PBS with 2% FCS; 5 mM EDTA), blocked with anti-CD16/32 Fc block (BD, Biosciences, Franklin Lakes, NJ, USA), and labeled with fluorochrome-conjugated antibodies for 1 h. Antibodies against CD45 (HI30; BD, Biosciences), CD3 (145-2C11; Invitrogen), CD4 (RM4-5; BioLegend), CD8 (53-6.7; BioLegend), Ly6C (HK1.4; BioLegend), Ly6G (1A8; BD, Bioscience), CD11b (M1/70; BioLegend) and NK1.1 (PK136; BD, Bioscience) were used, and a near infrared (NIR) LIVE/DEAD stain (Life Technology) was used to exclude dead cells. Counting beads (Spherobeads, BD) were added to samples before acquisition. Samples were acquired on a BD LSRFortessa flow cytometer, and data analysis was performed using FlowJo 10.7.
4
Quantifying Immune Cell Populations in Murine Lungs
5
Isolation of Inflammatory Leukocytes from CNS
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Inflammatory leukocytes infiltrating into the CNS were isolated using an established protocol (3,4). In short, CNS tissue was minced and leukocytes were isolated using a two‐step Percoll gradient (90% and 63%). The isolated cells were collected and then washed prior to staining. Cells were incubated in an anti‐CD16/32 Fc Block (BD Biosciences, San Jose, CA) at a dilution of 1:200. Cells were stained with fluorescently tagged rat anti‐mouse IgG for the following cell surface antibodies, Ly6G FITC (1A8), CD11b PE (M1/70), CD45 BV510 (30‐F11), CD8 PE‐Cy7 (53–6.7), (BD Biosciences), Ly6C APC (HK1.4), CD4 BV785 (RM4‐5), F4/80 FITC (BM8), I‐A/I‐E APC (M5/114.15.2), CD11c FITC (N418) (Biolegend, San Diego, CA), and CD4 FITC (RM4‐5) or Armenian hamster anti‐mouse IgG for CD80 PerCP‐Cy5.5 (16‐10A1) (BD Biosciences). A full description of gating strategies for flow cytometric staining and intracellular cytokine staining is provided in the Supporting Information Figs. S1–S4.
6
Characterization of T-cell Populations
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The cells were stained for surface and intracellular/intranuclear, and analysis by FACS Canto flow cytometer (BD Biosciences). The following fluorochrome conjugated Abs: PerCP Cy5.5-CD45, APC Cy7-CD45, PerCP Cy5.5-CD4, PE-CD4, FITC-CD4, FITC-CD3, APC-CD3e, PerCP-CD3e, APC Cy7-CD3, PE-CD11b,PE Cy7-CD11b, FITC-MHC-II, PerCP Cy5.5-MHCII, PE-CD19, FITC-CD19, PE Cy7-CD19, APC-CD25, PE Cy7-CD25, PerCp Cy5.5-CD25, APC-CD138, FITC-CD40, PE-FoxP3, FITC-IL10 and anti-CD16/32 (Fc block) were purchased from BD Pharmingen (San Diego, CA), Biolegend (San Diego, CA), or eBiosciences (Thermo Fisher Scientific, MA, USA). Frequencies and numbers of populations in the meninges, lymph nodes and spleens of B6 and BTBR mice were gated based on FSC-A and SSC-A, followed by gating in single cells and finally gated on CD45+ cells. Acquired FCS files were analyzed by Flow Jo-V10. Treg cell population were identified based on CD25+FoxP3+ cell in CD3+CD4+ population. To observe the Treg cell population, CD4+ cells were analyzed for the expression of CD25 and FoxP3 and gated on the double positive population. Data were obtained from three to four independent experiments with 4–6 pairs of mice in each experiment. In each experiment, equal number (2/3 males and 2/3 females from each group) of male and female mice were used.
7
Quantifying IgE+ Plasma Cells
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Mice were sacrificed at week 78 of the protocol and single-cell suspensions of splenocytes were prepared in ice cold staining buffer (PBS including 0.5 mM EDTA, 0.05 mM sodium azide, 0.5% BSA). First, surface staining with unlabeled anti-IgE (to block membrane IgE), APC anti-CD138, BV711-anti-CD3, and anti-CD16/32 (Fc-block) (all from BD Biosciences), CA was performed. Live-dead discriminating dye (Live-Dead Aqua, Invitrogen, CA) was included. Cells were washed and incubated with fixation/permeabilization buffer (BD Biosciences, CA) for 15 mins, washed with permeabilization buffer (BD Biosciences, CA), and then incubated with FITC-anti-IgE, in permeabilization buffer. After washing, cells were treated with Cytofix buffer (BD Biosciences, CA) for 15 mins for post-fixation, washed, and then data were acquired on an LSRII flow cytometer (Becton Dickinson, CA). Flow cytometry analysis was performed using Flow Jo (Tree Star, CA) as follows. Live singlet cells were then analyzed for IgE+ plasma cells (FITC-IgE +; APC-CD138+ cells).
8
Tumor Immune Cell Phenotyping
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Single cell suspensions were prepared as in the tumor dissociation protocol. Non-specific labeling was blocked using anti-CD16/32 (Fc block; BD Biosciences, Franklin Lakes, NJ) before specific labeling. BD Horizon fixable viability stain 780 was used to distinguish live and dead cells. Tumor cells were stained with antibodies targeting F4/80 PE, CD206 APC, and Ly6C Brilliant Violet 421 (all from BioLegend, San Diego, CA). Sample acquisition was performed with the BD LSR II cytometer and results analyzed with FlowJo software.
9
Multicolor Flow Cytometry for Immune Cell Analysis
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Blood was collected with heart puncture and transferred into a Na EDTA-containing tube. Single cell suspensions were prepared from spleens and BM as described (Heo, 2005 ). Whole blood (50 μl) was stained and erythrocytes were lysed with FACSlyse (BD Biosciences, San Jose) before analysis. Splenocytes and BM cells were counted, and 1 × 106 cells were used for staining followed by FACSlyse and analysis by six-color flow cytometry with a FACSCanto flow cytometer (BD Biosciences), and the following fluorochrome conjugated Abs: PerCP Cy5.5-CD45, APC Cy7-CD45, PerCP Cy5.5-CD4, PE-CD4, FITC-CD4, FITC-CD8a, PE-CD8a, PerCP-CD8a, FITC-CD3, APC-CD3e, PerCP-CD3e, PE Cy7-CD24, FITC-CD43, PerCP-CD249- APC Cy7-B220, PE -IgD, APC-IgM, APC Cy7-CD3, PE Cy7-CD19, PerCP cy5.5-CD93, APC-CD138, PE-CXCR4, FITC-MHC-II, PE-CD19, FITC-CD19, PE Cy7-CD19, APC-CXCR5, PE Cy7-PD1, PerCP Cy5.5-CD138 and anti-CD16/32 (Fc block). The Abs were purchased from BD Pharmingen (San Diego, CA), Biolegend (San Diego, CA), or eBiosciences (Thermo Fisher Scientific, MA, USA). Frequencies and numbers of populations in the blood and spleens of B6 and BTBR mice were gated based on FSC-A and SSC-A, followed by gating out the doublets and finally gated on CD45+ cell. Data were analyzed by Flow Jo-V10.
10
Macrophage Isolation and Characterization
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Naive macrophages were detached from non-tissue culture-treated plates after 7 days differentiation in M-CSF-containing media by scraping in versene. Cells were pelleted (1000 g , room temperature, 5 min), washed in FACS buffer [1% (w/v) bovine serum albumin in phosphate buffered saline (PBS)] and then kept at 4°C for subsequent steps. Cells were resuspended and incubated with 10 µg/ml anti-CD16/32 Fc block (BD Bioscience) in FACS buffer for 15 min, prior to incubation for 30 min with 1 µg/ml Brilliant Violet 421 anti-mouse F4/80 and 1 µg/ml PerCP/Cy5.5 anti-mouse/human CD11b (Biolegend) in FACS buffer. Cells were washed in FACS buffer and fixed by incubation with 4% formaldehyde in PBS for 20 min. Fixed cells were washed and resuspended in FACS buffer prior to analysis using FACS Canto (Becton Dickinson).
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