Ly6g bv650

Manufactured by BioLegend

The Ly6G BV650 product is a fluorochrome-conjugated antibody that can be used to identify and analyze cells expressing the Ly6G antigen. It is designed for flow cytometric applications.

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Anti-Ly6G-BV650 (3 citations)

3 protocols using ly6g bv650

Comprehensive Immune Cell Profiling in Mouse Lungs

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BMDCs and/or whole lung lysates were incubated with Zombie UV Fixable Viability Kit (BioLegend, San Diego, CA) at room temperature for 15 minutes. Cells were washed with FACS rinsing buffer, pelleted, and incubated with anti-mouse CD16/32 (BioLegend) for 15 minutes at 4 °C to block non-specific Ig binding. Following subsequent washing and centrifugation, cells were incubated with antibody cocktails containing some or all of the following markers: CD11c BV711, CD80 BV650, CD64 PerCP/Cy5.5, Ly6C BV605, CCR-7 APC, MHC-II (I-A/I-E) BV 421, Ly6G BV650, CD40 PE/Cy7,CD4 APC/Cy7, CD3 APC, and CD8α PerCP/Cy5.5 (BioLegend); CD86 BUV395, CD11b BV480, Siglec-F APC-R700, CD45 BV805, CD103 PE-CF594, and CD24 BUV 737 (BD Biosciences, San Jose, CA). Cells were analyzed using the BD LSRFortessa™ (BD Biosciences). Cell aggregates were removed by gating single cells on the forward light scatter (FSC-H vs FSC-A). Dead cell and debris were excluded before gating for myeloid innate immune cells and/or T-cell markers. Fluorescence minus one (FMO) controls were used to set up gating strategies used in these experiments. Data was analyzed using FlowJo software v10.7.1 (Tree Star Inc.).

Hall S.C., Smith D.R., Dyavar S.R., Wyatt T.A., Samuelson D.R., Bailey K.L, & Knoell D.L. (2021). Critical Role of Zinc Transporter (Zip8) in Myeloid Innate Immune Cell Function and the Host Response against Bacterial Pneumonia. Journal of immunology (Baltimore, Md. : 1950), 207(5), 1357-1370.

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Visualization of Neutrophil Extracellular Traps

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We employed an established technique using a combination of the cell-impermeable nucleic acid dye, SYTOX Green, and 4’,6-diamidino-2-phenylindole (DAPI), which is membrane-semi-permeable, for two-colour visualization of NETs on flow cytometry.^{75 (link),76 (link)} Single cell suspensions were obtained from nasal tissue and added to U-bottom 96-well polypropylene plates. Cells were incubated for 30 min at 37 °C in 5% CO₂ prior to stimulating with heat-killed B. pertussis (1 × 10⁷ CFU/mL), PMA (50 ng/mL; Sigma-Aldrich), or medium for 4 h. Cells were fixed by adding PFA at a final concertation of 1.3% and incubated for 15 min at RT. PFA concentrations below 4% are thought to fix, but not permeate the plasma membrane, thus reducing artificial “NET formation” .^{76 (link)} Samples were then centrifuged at 100 × g for 10 min at RT and supernatants were carefully aspirated. Samples were stained with the following: DAPI (0.1 µg/mL; Sigma-Aldrich), SYTOX Green (0.3 µM; Thermo-Fisher Scientific), CD11b-APCeFlour780 (MEL-14; eBioscience), Ly6G-BV650 (1A8; BioLegend), and Siglec-F-PECF594 (E50-2440; BD Biosciences).

Borkner L., Curham L.M., Wilk M.M., Moran B, & Mills K.H. (2021). IL-17 mediates protective immunity against nasal infection with Bordetella pertussis by mobilizing neutrophils, especially Siglec-F+ neutrophils. Mucosal Immunology, 14(5), 1183-1202.

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Comprehensive Immune Characterization of BALF

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BAL fluid was collected from each animal through cannulation of the exposed trachea, and flushing twice with .7mL of PBS. BALF samples were centrifuged at 500xG for 5 minutes, and supernatant was immediately frozen for later cytokine analyses. For flow cytometry, pelleted cells were resuspended in FACS (PBS + 5% FBS) buffer for staining and with Zombie NIR (Biolegend, 423105, SanDiego, CA), CD45, Alexa Fluor 532 (eBioscience, 58-0459-42,San Diego, CA), CD11c, PE-Cy7 (Biolegend, 117317, SanDiego, CA), CD11B, BV480 (BD Biosciences, 566117, SanJose, CA), SIGLEC F, AF700 (eBioscience, 56-1702-80, SanDiego, CA), Ly-6c, FITC (Biolegend, 128005, San Diego, CA),and Ly-6G, BV650 (Biolegend, 127641, San Diego, CA) and then fixed in IC-fixation buffer (eBioscience, 00-8222-49, San Diego, CA). Samples were run on a Cytek Aurora Borealis at the University of Virginia flowcytometry core. Neutrophils are Zombie NIR−, CD45+,CD11C−, CD11B+, and Ly-6G+; eosinophils are Zombie NIR−,CD45+, CD11C−, CD11B+, and Siglec-F+; inflammatory monocytes are Zombie NIR−, CD45+, CD11C−, CD11B+, Ly-6G−, and Ly-6C+.
Cytokine analyses were performed via Luminex Mouse 32-plex (MCYTMAG-70K-PX32, Millipore-sigma). Samples were run following manufacturers protocol after an 18-hour incubation before being run on Luminex® analyzer (MAGPIX®).

Donlan A.N., Sutherland T.E., Marie C., Preissner S., Bradley B.T., Carpenter R.M., Sturek J.M., Ma J.Z., Moreau G.B., Donowitz J.R., Buck G.A., Serrano M.G., Burgess S.L., Abhyankar M.M., Mura C., Bourne P.E., Preissner R., Young M.K., Lyons G.R., Loomba J.J., Ratcliffe S.J., Poulter M.D., Mathers A.J., Day A., Mann B.J., Allen J.E, & Petri WA J.r. (2021). IL-13 is a driver of COVID-19 severity. medRxiv.

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