Single cell suspensions of BAL cells and F4/80+ cells recovered from tissue digests (interstitial macrophages) were incubated with TruStain Fc block (Biolegend) for 10 min at 4 °C or 5 min at room temperature, respectively, to block nonspecific binding. BAL cells were then incubated with the following antibodies (1:100) for 30 min at 4 °C: F4/80-PE (Stem Cell), Cd11c-Af700 (Biolegend), Cd11b-FITC (Biolegend), Ly6G-Alexa Fluor 647 (Biolegend), Ly6C-PerCp5.5 (Biolegend), and then with eFluor 780-conjugated Fixable Viability Dye (Thermo Fisher). Cells were washed, fixed in 2% paraformaldehyde and analyzed on a Gallios flow cytometer (Beckman Coulter, Brea, CA); data were analyzed using Beckman Coulter’s Kaluza software. Following exclusion of doublets, viable BAL cells were analyzed for expression of F4/80 and CD11c. Double positive cells were then analyzed for expression of CD11b, Ly6G and Ly6C. Viable F4/80+ tissue cells were analyzed for expression of CD11b, followed by Ly6G, CD11c and Ly6C.
Ly6g alexa fluor 647
Manufactured by BioLegend
Sourced in United States
Ly6G-Alexa Fluor 647 is a fluorochrome-conjugated antibody that targets the Ly6G antigen. Ly6G is a cell surface marker expressed on granulocytes, including neutrophils. The Alexa Fluor 647 dye provides a far-red fluorescent signal that can be detected using flow cytometry and other fluorescence-based techniques.
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Lab products found in correlation
Cd11c af700, by BioLegend (1 mentions)
Cd11b fitc, by BioLegend (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Trustain fc block, by BioLegend (1 mentions)
Anti mouse cd16 32, by BioLegend (1 mentions)
Fc500 cytometer, by Beckman Coulter (1 mentions)
Cd11b pe cy7, by BioLegend (1 mentions)
Ly6c fitc, by BioLegend (1 mentions)
2 protocols using ly6g alexa fluor 647
1
Multiparametric Flow Cytometry of BAL and Tissue Macrophages
2
Quantifying MDSC Subsets and Activation
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Immune cells from at least 10 animals were counted by flow cytometry analysis using FC 500 cytometer (Beckman Coulter, USA). A total of 1×106 cells/100 μL were firstly incubated with anti-mouse CD16/32 (Biolegend, USA, 101320) to block non-specific staining then incubated with antibodies including CD11b - PE/Cy7, Ly-6G - Alexa Fluor 647 (BioLegend, USA, 127610), Ly-6C - FITC (Biolegend, USA, 128006) for MDSCs for 30 mins at 4°C.G-MDSC was defined as CD11b+ Ly6G+ Ly6Clo and M-MDSC was defined as CD11b+ Ly6G-Ly6Chi (3 (link)). The fixed cells were permeabilized in pre-chilled True-PhosTM Perm Buffer (Biolegend, USA, 425401) overnight at -20°C, then added intracellular staining antibody anti-STAT3 Phospho (Tyr705) - PE (Biolegend, USA, 651004) for detection of STAT3 Phospho expression as a marker of expansion and migration in MDSCs. The stained cells were washed and resuspended in 1mL staining buffer for further analysis.
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