Nasal lavages were pelleted at 1,500 rpm for 2 min and resuspended in 200 µl PBS containing 1% bovine serum albumin (BSA). After FcR blocking with a 1:200 dilution of anti-CD16/32 (BioLegend, San Diego, CA, USA) for 15 min, cells were stained for 30 min at 4°C with 25 µl of 1:150 dilution of the following antibodies: anti-CD11b-V450 (BD Biosciences, San Jose, CA, USA), anti-F4/80-PE (BioLegend), anti-Ly6G-PerCP-Cy (BD Biosciences), and anti-CD45-APC-Cy7 (BD Biosciences) for 30 min on ice in the dark. Cells were washed with PBS with 1% BSA, then fixed with 4% PFA. FACSVerse (BD) was used for flow cytometry analysis. After excluding dead cells and debris by gating with forward and side scatter, neutrophils (polymorphonuclear cells; PMNs) were detected as CD11b+, Ly6G+, and CD45+ components. Macrophages were detected as F4/80+, Ly6G-, and CD45+ components. The absolute number of cells in each sample was counted.
Anti ly6g percp cy
Manufactured by BD
Sourced in United States
The Anti-Ly6G-PerCP-Cy is a fluorochrome-conjugated monoclonal antibody designed for the detection and quantification of Ly6G-positive cells, such as neutrophils, in flow cytometry applications. It binds specifically to the Ly6G surface antigen, which is expressed on neutrophils. The PerCP-Cy fluorescent label allows for the identification and enumeration of Ly6G-expressing cells within a sample.
Automatically generated - may contain errors
3 protocols using anti ly6g percp cy
1
Flow Cytometric Quantification of Immune Cells
2
Neutrophil Quantification in Nasal Lavage
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Neutrophils in nasal lavages were stained as previously described (Kono et al., 2016 (link); Zafar et al., 2017 (link)). Pups exposed to CSE or PBS were pre-treated and infected with 8,000 CFU Sp. as described above. Pups were euthanized on day 2 or 4 p.i. and nasal lavages (200 µl) were collected. Pellets of nasal lavages were resuspended with PBS + 1% bovine serum albumin. Cells were stained with a 1:150 dilution of the following antibodies: anti-CD11b-V450 (BD), anti-Ly6G-PerCP-Cy (BD), and anti-CD45-APC-Cy7 (BD) for 30 min on ice in the dark after FcR blocking with a 1:200 dilution of anti-CD16/32 (BioLegend) for 15 min on ice. Cells were then fixed with 4% paraformaldehyde until analysis on FACS Verse (BD). Neutrophils were detected as CD11b+, Ly-6G+, CD45+ events.
3
Quantification of Nasal Immune Cells
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Neutrophil and macrophage cell counts were performed as previously described [28 (link), 31 (link)]. The nasal lavages were
pelleted at 1,500 rpm for 2 min, and resuspended in 200 µl PBS containing 1% bovine serum albumin (BSA). After FcR blocking with a 1:200 dilution of anti-CD16/32 (BioLegend,
San Diego, CA, USA) for 15 min, cells were stained for 30 min at 4°C with 25 µl of 1:150 dilution of the following antibodies: anti-CD11b-V450 (BD Biosciences, San Jose, CA,
USA), anti-F4/80-PE (BioLegend), anti-Ly6G-PerCP-Cy (BD Biosciences), and anti-CD45-APC-Cy7 (BD Biosciences) for 30 min on ice in the dark. Cells were washed with PBS with 1% BSA, then fixed
with 4% PFA. FACS Verse (BD) was used for flow cytometry analysis. After excluding dead cells and debris by gating with forward and side scatter, neutrophils (polymorphonuclear cells; PMNs)
were detected as CD11b+, Ly6G+, and CD45+ components. Macrophages were detected as F4/80+, Ly6G−, and CD45+ components
(Supplementary Fig. 1 ). The absolute number of cells in each sample were counted.
pelleted at 1,500 rpm for 2 min, and resuspended in 200 µl PBS containing 1% bovine serum albumin (BSA). After FcR blocking with a 1:200 dilution of anti-CD16/32 (BioLegend,
San Diego, CA, USA) for 15 min, cells were stained for 30 min at 4°C with 25 µl of 1:150 dilution of the following antibodies: anti-CD11b-V450 (BD Biosciences, San Jose, CA,
USA), anti-F4/80-PE (BioLegend), anti-Ly6G-PerCP-Cy (BD Biosciences), and anti-CD45-APC-Cy7 (BD Biosciences) for 30 min on ice in the dark. Cells were washed with PBS with 1% BSA, then fixed
with 4% PFA. FACS Verse (BD) was used for flow cytometry analysis. After excluding dead cells and debris by gating with forward and side scatter, neutrophils (polymorphonuclear cells; PMNs)
were detected as CD11b+, Ly6G+, and CD45+ components. Macrophages were detected as F4/80+, Ly6G−, and CD45+ components
(
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