Cells were stained with BD Biosciences (Oxford, U.K.) mAbs: CD45-PerCP-Cy5.5 (30-F11), CD45.2-V450 (104), CD4-BV650 (RM4-5), CD3-FITC (17A2), CD11b-eFluor450 (M1/70), CD19-BV711 (1D3), SiglecF (E50-2440), CD103-PE-CF594 (M290), Ly6G-BV650 (1A8); eBioscience (Loughborough, U.K.) mAbs: CD4-allophycocyanin-eFluor780 (RM4-5); Invitrogen (Dublin, Ireland) mAbs: KLRG1-PE-eFluor610 (2F1) and CD127-PerCP-ef710 (SB/199); and BioLegend (London, U.K.) mAbs: CD45-BV711 (clone: 30-F11), CD3-BV605 (17A2), CD11b-allophycocyanin-Cy7 (M1/70), CD11c-PE-Cy7 (N418), Ly6G-BV785 (1A8), Ly6C-BV606 (HK1.4), and SiglecF-allophycocyanin (S1700L). Before surface staining, Fc receptors were blocked using Fc-Block CD16/32 (BD Biosciences), and cells were incubated with LIVE/DEAD Fixable Aqua stain (Molecular Probes, Invitrogen) to isolate dead cells. For staining of transcription factors, cells were fixed and permeabilized using the Foxp3 staining buffer kit (Invitrogen) and stained with mAbs: GATA3-PE (TWAJ) and Foxp3-PE-Cy7 (FJK-16s). For the detection of YFP, along with intracellular transcription factors from Rora-YFP mice, after surface markers and viability stain, cells were prefixed with 2% paraformaldehyde followed by Foxp3 staining buffer kit. Cells were analyzed using a BD Fortessa (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR), using appropriate controls.
Cd16 32 fc block
Manufactured by BD
Sourced in United Kingdom
The CD16/32 (Fc block) is a laboratory reagent used in flow cytometry and cell-based assays. Its core function is to block Fc receptors, which can non-specifically bind to antibodies and interfere with the specific target binding. This product helps to minimize background signal and improve the accuracy of flow cytometry and cell-based experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (3 mentions)
Anti b220 ra3 6b2, by Thermo Fisher Scientific (3 mentions)
Gata 3 pe, by Thermo Fisher Scientific (2 mentions)
Foxp3 pe cy7, by Thermo Fisher Scientific (2 mentions)
Cd3 bv605, by BioLegend (2 mentions)
Cd11c pe cy7, by BioLegend (2 mentions)
Fortessa, by BD (2 mentions)
Live dead fixable aqua stain, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining buffer kit, by Thermo Fisher Scientific (2 mentions)
Siglecf e50 2440, by Thermo Fisher Scientific (2 mentions)
Ly6g bv785, by BioLegend (2 mentions)
Cd11b efluor450 m1 70, by Thermo Fisher Scientific (2 mentions)
Cd19 bv711 1d3, by Thermo Fisher Scientific (2 mentions)
Cd4 allophycocyanin efluor780 rm4 5, by Thermo Fisher Scientific (2 mentions)
Klrg1 pe efluor610 2f1, by BioLegend (2 mentions)
Cd127 percp ef710 sb 199, by BioLegend (2 mentions)
Cd11b allophycocyanin cy7, by BioLegend (2 mentions)
Ly6c bv606, by BioLegend (2 mentions)
Cd3 fitc 17a2, by Thermo Fisher Scientific (2 mentions)
Cd45 bv711, by BioLegend (2 mentions)
21 protocols using cd16 32 fc block
1
Flow Cytometry Immunophenotyping
2
Multi-marker Profiling of Immune Cells
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Isolated SVF cells or leukocytes were resuspended in 200 µl PBS containing 0.25% BSA, 0.2 mM EDTA, and 1% penicillin/streptomycin. Cells were preincubated for 7 min at 4 °C in Fc Block (CD16/32, BD Biosciences) and then stained for 15 min with fluorophore-conjugated antibodies at 4 °C. The following antibodies were used: anti-CD45 (clone: 30-F11, Biolegend), anti-F4/80 (clone: BM8, Biolegend), anti-CD11b (clone: M1/70, Biolegend), anti-CD11c (clone: N418, Biolegend), anti-CD206 (clone: C068C2, Biolegend), anti-CCR2 (clone: SA203G11, Biolegend), anti-Ly6G/Ly6C (clone: RB6-8C5, Biolegend), and anti-CD115 (clone: AFS98, Biolegend)15 (link). Flow cytometric analysis was performed using a FACSCantoII (BD Biosciences). Cell sorting was performed with a FACSAriaII (BD Biosciences). Data were analyzed using FlowJo software (v9.4.10, Tree Star).
3
Multiparameter Flow Cytometry of BALF
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BALF was collected from control and IAV infected WT and IFNγR-/- mice. Lungs were flushed multiple times with 1 ml ice-cold sterile HBSS with 3 mM EDTA, pH 7.2. BALF was pooled and maintained on ice until centrifugation at 350×g for 10 min, 4 °C, and treated with red blood cell lysis buffer prior to further analysis. Cells were resuspended in FACS buffer (PBS/FCS) with Fc block (CD16/32, 0.5 mg/ml; BD Biosciences, UK) and stained for 1 h at 4 °C using the following antibodies: anti-CD11b-APC (M1/70.11.5; Miltenyi Biotec Ltd., UK), anti-Ly6G-PE/Cy7 (BD Biosciences), anti-CD4-PE (RM4–5; BD Biosciences), anti-CD8-FITC (53–6.7; BD Biosciences), anti-CD11c-PE/Cy7 (N418; BioLegend, UK), anti-Siglec-F BV421 (E50–2440; BD Biosciences), and appropriate isotype/FMO controls. Cell viability was determined using Zombie fixable dyes during staining to exclude dead cells (Zombie Aqua or Violet; BioLegend). Cells were fixed with BD Cytofix (BD Biosciences) and analysed using the BD LSR Fortessa and FlowJo V10 software (FlowJo LLC, USA) using a gating strategy adapted from Misharin et al. (2013) (link) (Supplementary Fig. S4 ).
4
Multiparameter Flow Cytometry Analysis
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Cells were incubated with Fc Block (CD16/32, BD Biosciences, San Jose, CA), stained with antibodies, and then fixed with 2% PFA. Samples were acquired on the FACS Canto II (BD Biosciences) and analyzed using FlowJo (TreeStar, Ashland, OR). Dead cells were excluded using the eBioscience Fixable Viability Dye eFluor® 506 (Thermo Fisher Scientific, Waltham, MA). The following antibodies were used: anti-CD8 (53-6.7) and anti-TCR-γδ (ebioGL3) from Thermo Fisher Scientific; anti-CD4 (GK1.5), anti-CD19 (6D5), anti-CD11b (M1/70), and anti-CD45 (30-F11) from BioLegend, San Diego, CA; and anti-TCR-β (H57-597) from BD Biosciences. APC-conjugated mouse CD1d tetramers loaded with glycolipid PBS-57 (CD1d-tet) and an unloaded tetramer comprised of only the glycolipid PBS57 were obtained from the tetramer facility of the National Institutes of Health (NIH).
5
Flow Cytometry Immunophenotyping
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Cells were stained with BD Biosciences (Oxford, U.K.) mAbs: CD45-PerCP-Cy5.5 (30-F11), CD45.2-V450 (104), CD4-BV650 (RM4-5), CD3-FITC (17A2), CD11b-eFluor450 (M1/70), CD19-BV711 (1D3), SiglecF (E50-2440), CD103-PE-CF594 (M290), Ly6G-BV650 (1A8); eBioscience (Loughborough, U.K.) mAbs: CD4-allophycocyanin-eFluor780 (RM4-5); Invitrogen (Dublin, Ireland) mAbs: KLRG1-PE-eFluor610 (2F1) and CD127-PerCP-ef710 (SB/199); and BioLegend (London, U.K.) mAbs: CD45-BV711 (clone: 30-F11), CD3-BV605 (17A2), CD11b-allophycocyanin-Cy7 (M1/70), CD11c-PE-Cy7 (N418), Ly6G-BV785 (1A8), Ly6C-BV606 (HK1.4), and SiglecF-allophycocyanin (S1700L). Before surface staining, Fc receptors were blocked using Fc-Block CD16/32 (BD Biosciences), and cells were incubated with LIVE/DEAD Fixable Aqua stain (Molecular Probes, Invitrogen) to isolate dead cells. For staining of transcription factors, cells were fixed and permeabilized using the Foxp3 staining buffer kit (Invitrogen) and stained with mAbs: GATA3-PE (TWAJ) and Foxp3-PE-Cy7 (FJK-16s). For the detection of YFP, along with intracellular transcription factors from Rora-YFP mice, after surface markers and viability stain, cells were prefixed with 2% paraformaldehyde followed by Foxp3 staining buffer kit. Cells were analyzed using a BD Fortessa (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR), using appropriate controls.
6
Lung Cell Cytokine Profiling and Antibody Neutralization
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Lung cells were rst blocked for Fc receptors with Fc Block (CD16/32, BD Bioscience) and then surfacestained with uorescein-conjugated antibodies in PBS for 30 min at 4 °C under light protection. For intracellular cytokine staining, naïve CD4 + T cells were isolated from the lungs of mice and stimulated with PMA (25 ng/mL, Sigma) and ionomycin (1 μg/mL, Sigma) for 4.5 h at 37 °C. Brefeldin A (10 μg/mL, Sigma) was added for the last 4 h of culture. After incubation, intracellular staining was performed according to the manufacturer's instructions (BD Pharmingen). Antibodies used were as follows: anti-IFNγ-PE (Biolegend), anti-MHC II-APC/cy7 (Biolegend), anti-CD80-BV421 (Biolegend), anti-CD86-PE/cy7 (Biolegend), anti-CD3-FITC (Biolegend), anti-CD4-PerCP (Biolegend), anti-CD69-APC/cy7 (Biolegend), anti-IL-4-PE (Biolegend), anti-IL-5-BV421 (eBioscience), anti-IL-13-PE/cy7 (eBioscience), anti-CD45-APC (eBioscience), anti-ST2-PE (eBioscience), and FITC-conjugated anti-lineage antibodies (eBioscience). The lineage marker antibody cocktail was composed of antibodies against: DX5 (or NK1.1), CD3, CD4, CD5, CD8, CD11b, CD19, B220, Gr-1 and TCRδ (BD Bioscience).
Neutralizing Antibody: Anti-mouse MHC Class II (clone: M5/114; InVivoMAb grade; BioXcell, BE0108), anti-mouse CD4 (α-CD4; clone: GK1.5; InVivoMAb grade; BioXcell, BE0003).
Neutralizing Antibody: Anti-mouse MHC Class II (clone: M5/114; InVivoMAb grade; BioXcell, BE0108), anti-mouse CD4 (α-CD4; clone: GK1.5; InVivoMAb grade; BioXcell, BE0003).
7
Multiparametric Analysis of Tumor-Infiltrating Lymphocytes
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Single cell suspension of TILs and spleen was stained with the live dead exclusion stain ef506 (eBioscience). The cells were subsequently incubated in Fc block (CD16/32) (BD Biosciences) for 30 min before proceeding with surface staining with fluorescently conjugated antibodies (Supplementary Table S1
8
Brain and Spinal Cord Cell Isolation
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Mice were transcardially perfused with PBS before tissue extraction. Either whole brains, spinal cords, or specific parts of the brain (cortex or cerebellum), not excluding meninges and choroid plexus, were collected, as indicated in the figure legends. Single-cell suspensions of tissues for all mice, excluding the Trem2 experiment, were achieved using software-controlled sealed homogenization system (Dispomix; http://www.biocellisolation.com) in PBS, followed by density gradient separation; Pellet was mixed with 40% percoll and centrifuged in 800G for 20 min at 12 C. Brains of Trem2 mice were processed as previously published (Wang et al., 2015) . Supernatant was discarded and pellet was taken further for antibody staining, as described below. Before staining, samples were blocked with Fc-block CD16/32 (BD Biosciences, San Jose, CA).
9
Multiparametric Flow Cytometry Analysis
10
Flow Cytometric Analysis of Lymphocyte Activation and Cytokine Production
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Prior to staining, cells were blocked with CD16/32 (Fc Block; BD). For surface staining, antibodies against the surface markers CD4, CD8, CD3, CD69, CD11b, CD11c, MHC class II (MHC-II), CD27, and DX5 (BD) were added at a 1:100 dilution for 30 min on ice. Gating for lymphocytes was determined by back gating on CD3/CD8 double-positive cells. For the detection of intracellular cytokines, cells were incubated with 50 ng/ml phorbol myristate acetate, 500 ng/ml ionomycin, and 10 μg/ml brefeldin A for 4 h at 37°C. Samples were permeabilized with 0.5% saponin in phosphate-buffered saline (PBS) for 10 min. Anticytokine antibodies (anti-IFN-γ, anti-IL-4; BD) or isotype controls were added for a further 20 min at room temperature. Cells were analyzed on a CyAn ADP flow cytometer (Dako), with data on at least 50,000 events being collected.
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