For ImageStream analysis, the following antibodies were used: Thermo Fisher; Fixable Viability Dye eFluor‐780 (catalogue number 65‐0865‐14), anti‐CD16/CD32 (clone 93), CD3‐APC‐ef780 (clone 145‐2C1), NKp46‐APC‐ef780 (clone 29A1.4), CD19‐APC‐ef780 (clone HIB19), F4/80‐eFluor 506 (clone AG BM8), CD11b‐PE (clone M1/70; BD), Siglec‐F‐SB645 (clone E50‐2440; Biolegend) and Ly6G‐PerCPCy5.5 (clone 1A8). Data acquisition was performed on ImageStream X (Amnis/EMD Millipore, Seattle, WA) running INSPIRE (version 200.1.681.0). Images of cells were acquired with a 60× objective. Eosinophils were identified as Siglec‐F+F4/80int and macrophages as Siglec‐F−F4/80hi events after removal of lineage‐positive events (CD3, CD19, NKp46), dead cells, and neutrophils (Ly6Ghi Siglec‐F− F4/80−). All data analysis was performed using the IDEAS® software version 6.
Ly6g percpcy5.5 clone 1a8
Manufactured by BioLegend
Ly6G-PerCPCy5.5 (clone 1A8) is a monoclonal antibody that binds to the Ly6G antigen. Ly6G is a marker for neutrophils in mice. The antibody is conjugated with the PerCPCy5.5 fluorochrome, which can be detected using flow cytometry.
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Lab products found in correlation
Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
Cd11b pe clone m1 70, by BD (1 mentions)
Anti cd16 cd32 clone 93, by BD (1 mentions)
Lrs fortessa x 20, by BD (1 mentions)
Cd3 apc, by BioLegend (1 mentions)
Cd8 bv711, by BioLegend (1 mentions)
F4 80 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd4 bv605, by BioLegend (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
Nk1.1 pe, by BioLegend (1 mentions)
Cd11b alexa700, by BioLegend (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Cd11b fitc clone m1 70, by BioLegend (1 mentions)
Ccr2 pe clone 475301, by R&D Systems (1 mentions)
Lsrii fortessa, by BD (1 mentions)
Apc cy7, by BioLegend (1 mentions)
4 protocols using ly6g percpcy5.5 clone 1a8
1
Multiparametric ImageStream Analysis
2
Multi-color Flow Cytometry Immunophenotyping
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For direct multi-colour flow cytometry (LRS Fortessa X-20; BD Bioscience), cells were incubated for 30 min at 4 °C with the antibodies mentioned below. Live/dead cell discrimination was performed by staining with the Zombie Aqua Fixable Viability Kit (BioLegend, catalogue number: 423101). To block Fc receptors, a purified anti-mouse CD16/CD32 (eBioscience, catalogue number: 14-0161-86) was used.
CD19 APCCy7 (clone 6D5, Biolegend, catalogue number: 115530, 1:300), Ly6G PerCPCy5.5 (clone 1A8, Biolegend, catalogue number: 127616, 1:300), CD45 PEDazzle (clone 30-F11, Biolegend, catalogue number: 103146, 1:400), F4/80PECy7 (clone BM8, eBioscience, catalogue number:254801-82, 1:200), CD3 APC (clone 145-2c11, Biolegend, catalogue number: 100312, 1:100), CD11b Alexa700 (clone M170, Biolegend, catalogue number: 101222, 1:400), TCγδV421 (clone GL3, Biolegend, catalogue number: 118120, 1:100), CD4 BV605 (clone RM4-5, Biolegend, catalogue number: 100548, 1:300), CD8 BV711 (clone 53-6.7, Biolegend, catalogue number: 100748, 1:300) and NK1.1 PE (clone PK136, Biolegend, catalogue number: 557391, 1:300). IL-10GFP production was measured in the FITC channel.
CD19 APCCy7 (clone 6D5, Biolegend, catalogue number: 115530, 1:300), Ly6G PerCPCy5.5 (clone 1A8, Biolegend, catalogue number: 127616, 1:300), CD45 PEDazzle (clone 30-F11, Biolegend, catalogue number: 103146, 1:400), F4/80PECy7 (clone BM8, eBioscience, catalogue number:254801-82, 1:200), CD3 APC (clone 145-2c11, Biolegend, catalogue number: 100312, 1:100), CD11b Alexa700 (clone M170, Biolegend, catalogue number: 101222, 1:400), TCγδV421 (clone GL3, Biolegend, catalogue number: 118120, 1:100), CD4 BV605 (clone RM4-5, Biolegend, catalogue number: 100548, 1:300), CD8 BV711 (clone 53-6.7, Biolegend, catalogue number: 100748, 1:300) and NK1.1 PE (clone PK136, Biolegend, catalogue number: 557391, 1:300). IL-10GFP production was measured in the FITC channel.
3
Flow Cytometric Analysis of Leukocytes During Decompression Illness
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Repeated blood sampling of mice was performed via saphenous vein puncture without anesthesia. Blood samples were collected using a 23G needle with ethylenediaminetetraacetic acid (EDTA)-coated tubes to avoid blood coagulation [15 (link)] and were analyzed by flow cytometry [16 ]. Samples were collected at 12 weeks of DCM, and at 24 h, 2 weeks, and 5 weeks after decompression. Red blood cells where lysed in red blood cell lysis buffer [7 ] and washed twice with phosphate-buffered saline (PBS). Cells were first stained with viability dye (Fixable viability dye eFluor 780, Thermo Fisher Scientific) for 20 min, followed by extracellular staining with fluorescent antibodies to distinguish granulocytes, monocytes, and T cells in the blood. The antibodies used were as follows: Ly6C-Pacific blue (clone HK1.4; BioLegend), Ly6G-PerCP/Cy5.5 (clone 1A8; BioLegend), CD11b- FITC (clone M1/70; BioLegend), CD3-PE (clone 17A2; BioLegend), and CCR2-PE (clone 475301; R&D Systems). Matching isotype controls were used to set the gates during data acquisition and analysis. Data were acquired using a BD LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo X 10 (Trestar).
4
Comprehensive Flow Cytometry Analysis of Lung Immune Cells
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Single cell suspensions were stained for flow cytometric analysis. Fc receptors were blocked using human-γ globulin, followed by surface marker staining with antimouse antibodies: CD11c (eFluor450, clone N418, eBioscience), CD11b (Alexa700, clone M1/70, BD Biosciences), Ly6G (PerCp-Cy5.5, clone 1A8, Biolegend), Ly6C (APC, clone HK1.4, eBioscience), F4/80 (PE, clone BM8, eBioscience), CD3e (PE-Cy7, clone 145–2C11, BD Biosciences or BV785 Biolegend), CD4 (PE-Cy5, clone RM4-5, BD Biosciences), CD8a (BV605, clone 53–6.7, BD Biosciences), CD49b (APC-Cy7, Biolegend or APC-e780, clone DX5, Affymetrix Inc), and MHC-II (FITC, clone M5/114.15.2, eBioscience). Samples were run on a LSRII Fortessa (Becton Dickinson, San Jose, CA) and data were analyzed using FlowJo 10.0.8 (Tree Star, Ashland, OR). Viable cells were gated from a forward scatter/side scatter plot and singlet inclusion. Following neutrophil exclusion (Ly6Ghi), macrophages (MΦ) were gated as CD11chiF4/80hi with alveolar macrophages (AMΦ) subgated as CD11b– and inflammatory macrophages (iMacs) as CD11b+. AMΦ were confirmed to have high side-scatter and be negative for MHC-II, CD11b, CD3e, CD4, CD8a, and DX5. The absolute numbers of cells were calculated based on the gating of viable events by flow cytometry and normalized to the total number of viable cells in the lung digest.
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