Blood samples (100 μL) from naïve or challenged mice were treated with RBC lysis buffer (Biolegend), centrifuged (350 × g) and supernatants discarded. Cells in pellets were washed in cold PBS and resuspended in PBS with 5% BSA for staining with anti-CD11b-APC/Cy7 (Biolegend), anti-Ly6G-BV650 (Biolegend), anti-FcγRI-PerCPCy5.5 (Biolegend), anti-FcγRIIb-PE (Biolegend), anti-FcγRIII-PE/Cy7 (Biolegend), anti-FcγRIV-FITC (Biolegend), and anti-CD11c-APC (Biolegend) antibodies in the dark and on ice for 30 min. All stained samples were analyzed using a flow cytometer (BD LSRII 3-8, BD Biosciences). Total neutrophils were obtained by gating CD11b+Ly6G+ cells. The fluorescence intensity of each receptor in neutrophils resulted in a single peak, thus the median fluorescence intensity (MFI) was used to represent the level of these receptors. 100% was set as the MFI data recorded for each receptor from PBS-treated BALB/cJ mice. Experiments were performed at least twice.
Anti ly6g bv650
Manufactured by BioLegend
The Anti-Ly6G-BV650 is a fluorochrome-conjugated antibody that binds to the Ly6G antigen, which is expressed on the surface of neutrophils. It is designed for use in flow cytometry applications to identify and quantify neutrophils in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b apc cy7, by BioLegend (2 mentions)
Anti cd11c apc, by BioLegend (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Anti cd45 bv711, by BioLegend (1 mentions)
Anti cd11c pe, by BD (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Anti f4 80, by Thermo Fisher Scientific (1 mentions)
Anti ly6g, by Thermo Fisher Scientific (1 mentions)
Anti cd8, by Thermo Fisher Scientific (1 mentions)
Anti b220, by Thermo Fisher Scientific (1 mentions)
Anti siglec f, by Miltenyi Biotec (1 mentions)
Anti ifn γ bv510, by BioLegend (1 mentions)
Anti siglecf pe, by BD (1 mentions)
Anti cd4, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti ly6g bv650
1
Quantifying Neutrophil Fc Receptor Expression
2
Flow Cytometric Analysis of Immune Cells
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Cells recovered from air pouches were counted and resuspended in block containing anti-mouse CD16/CD32. Cells were stained anti-Siglec F-APC (Clone: 1RNM44N, Invitrogen), anti-CD11b-APC-Cy7 (Clone: M1/70, Biolegend), anti-CD3/anti-CD19-BV421 (Clone CD3: 17A2, Biolegend; Clone CD19: eBio1D3 (1D3), Invitrogen), anti-Ly6G-BV650 (Clone 1A8, Biolegend), anti-CD45-BV711 (Clone: 30-F11, Biolegend) and anti-CD11c-PE (Clone: HL3, BD Pharmingen). A live/dead marker (Live/Dead Aqua; Thermo Scientific) was included to ensure analysis of viable cells only. Cells were acquired using a BD LSR Fortessa flow cytometer (BD Biosciences) followed by analysis with FlowJo software.
3
Flow Cytometric Analysis of Innate Immune Cells
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Proportions of granulocytes (eosinophils, neutrophils) populations were determined by flow cytometry using anti-SiglecF-PE (BD, clone E50-2440) and anti-Ly6G-BV650 (Biolegend, clone 1A8) rat anti-mouse antibodies. For innate lymphoid cell populations characterization, a lineage cocktail comprised of anti-CD8 (eBioscience, clone 53-6.7), anti-B220 (eBioscience, clone RA3-6B2), anti-F4/80 (eBioscience, clone BM8), anti-SiglecF (Miltenyi Biotec, clone ES22-10D8), anti-CD4 (eBioscience, clone GK1.5), anti-Ly6G (eBioscience, clone RB6-8C5) and anti- FcƐR1 (eBioscience, clone MAR-1) antibodies conjugated to APC was used in combination with anti-ST2-PE (eBioscience, clone RMST2-2), anti-NKp46-AF700 (BD, clone 29A1.4), anti- IL-12Rβ2-AF488 (R&D, clone 305719) and anti-CD127-PerCPCy5.5 (eBioscience, clone A7R34). In addition, NK cell activation was investigated using additional anti-IFNγ- BV510 (clone XMG1.2, Biolegend) and anti-Ly6C-BV785 (clone HK1.4, Biolegend) antibodies. All panels included a viability staining using the eF450 viability dye (Invitrogen) and all antibodies were used at a 1/40 dilution. Representative gating strategy is given in Supplementary Figure 1
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