Anti ly6g biotin

Manufactured by Miltenyi Biotec

Sourced in United States

Anti-Ly6G biotin is a biotinylated monoclonal antibody that specifically binds to the Ly6G antigen. Ly6G is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the surface of murine neutrophils. The antibody can be used for the detection and isolation of murine neutrophils.

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Lab products found in correlation

Streptavidin beads, by Miltenyi Biotec (3 mentions) Lineage depletion kit, by Miltenyi Biotec (3 mentions) Anti biotin microbead, by Miltenyi Biotec (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Gm csf, by Thermo Fisher Scientific (1 mentions) Recombinant g csf, by Thermo Fisher Scientific (1 mentions) Ly2228820 p38 inhibitor, by Selleck Chemicals (1 mentions) Recombinant gm csf, by Thermo Fisher Scientific (1 mentions) Cd34 microbead kit, by Miltenyi Biotec (1 mentions) Anti cd45r b220 biotin, by Miltenyi Biotec (1 mentions) Anti cd11c pe, by Miltenyi Biotec (1 mentions) Bms ccr2 22, by Bio-Techne (1 mentions) Anti cd11b bv421, by BD (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Raw264.7 cell, by American Type Culture Collection (1 mentions) Collagenase 2, by Worthington (1 mentions) Thioglycollate, by BD (1 mentions) Anti biotin magnetic beads, by Miltenyi Biotec (1 mentions)

7 protocols using anti ly6g biotin

Inflammatory Monocyte Stimulation by Aspergillus

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Biogel-elicited cells were washed with MACS buffer, PBS that was supplemented with 0.5% BSA and 2 mM EDTA, and stained with anti-Ly6G biotin (Miltenyi Biotec). Cells were incubated with anti-biotin microbeads (Miltenyi Biotec), and inflammatory monocytes (Ly6G⁻ cells) were enriched by MACS separation according to manufacturer’s instructions. Cells were resuspended in assay buffer, and 2–4 × 10⁵ cells were plated in a 96-well ultra-low adherence plate in 25 µl assay buffer. Assay buffer, 4 µM NE, or HI NE (25 μl) was added for 1 h. A. fumigatus SC (0.6–1.2 × 10⁶) in 50 µl Aim V medium was added for 4 h to stimulate cells at a 3:1 A. fumigatus:cell ratio. NE and HI NE remained in the cultures during stimulation with A. fumigatus. Cell culture supernatants were recovered and assayed for cytokine by ELISA according to manufacturer’s protocol.

Griffiths J.S., Thompson A., Stott M., Benny A., Lewis N.A., Taylor P.R., Forton J., Herrick S., Orr S.J, & McGreal E.P. (2018). Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases. The FASEB Journal, 32(6), 3385-3397.

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Generation of Mouse Neutrophils from BM

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Mouse neutrophils were generated from enriched bone marrow hematopoietic progenitor cells (HPCs) with 20 ng/ml of GM-CSF. Briefly, HPCs were isolated from mouse BM by using Lineage depletion kit (Miltenyi), according to manufacturer’s instructions. Cells were seeded at 25000 cell/ml in 24 well plates and GM-CSF (20 ng/ml), 20% v/v TES or arachidonic acid (10μM) were added at day 0 and day 3. At day 5, Ly6G positive neutrophils were isolated by using anti-Ly6G biotin (Miltenyi) and streptavidin beads (Miltenyi), according to manufacturer’s instructions.

Veglia F., Tyurin V.A., Blasi M., De Leo A., Kossenkov A., Donthireddy L., Jerrick T.T., Schug Z., Basu S., Wang F., Ricciotti E., DiRusso C., Murphy M.E., Vonderheide R.H., Lieberman P.M., Mulligan C., Nam B., Hockstein N., Masters G., Guarino M., Lin C., Nefedova Y., Black P., Kagan V.E, & Gabrilovich D. (2019). Fatty acid transporter 2 reprograms neutrophils in cancer. Nature, 569(7754), 73-78.

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Hematopoietic Progenitor Cell Isolation and Characterization

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Mouse. Hematopoietic progenitor cells (HPCs) were isolated from mouse bone marrow by using a Lineage depletion kit (Miltenyi), according to the manufacturer’s instructions. Cells were seeded at 25,000 cell/ml in 24-well plates and recombinant GM-CSF (20 ng/ml; Invitrogen), 20% v/v TES were added on day 1 and day 3. At day 5, Ly6G positive neutrophils were isolated by using anti-Ly6G biotin (Miltenyi) and streptavidin beads (Miltenyi), according to manufacturer’s followed by suppression assay. In addition, total cells were stained and analyzed for flow cytometry. In other experiments, LY2228820 p38 inhibitor (1 μM; Selleckchem) was added to HPC culture on day 3.
Human. Hematopoietic progenitor cells (HPCs) were isolated from cord blood using the CD34 MicroBead Kit (Miltenyi), according to the manufacturer’s instructions. Cells were seeded at 5 × 10⁴ cell/ml in 6-well plates with recombinant G-CSF (100 ng/ml; PeproTech) and GM-CSF (10 ng/ml; PeproTech). At day 7, 30% v/v TES were added and the next day flow cytometry analysis was performed.

Alicea-Torres K., Sanseviero E., Gui J., Chen J., Veglia F., Yu Q., Donthireddy L., Kossenkov A., Lin C., Fu S., Mulligan C., Nam B., Masters G., Denstman F., Bennett J., Hockstein N., Rynda-Apple A., Nefedova Y., Fuchs S.Y, & Gabrilovich D.I. (2021). Immune suppressive activity of myeloid-derived suppressor cells in cancer requires inactivation of the type I interferon pathway. Nature Communications, 12, 1717.

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Isolation of Peritoneal and Alveolar Macrophages

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Peritoneal macrophages were induced by intraperitoneal injection of 1 ml of sterile liquid paraffin. After 5 days, macrophages were collected through peritoneal lavage with 5 ml cold PBS. Alveolar macrophages were collected by inflating five times with 1 ml cold PBS via an intratracheal cannula and was repeated a further two times. Macrophages were isolated by adherence 40 min on culture plastic. Neutrophils were purified from the bone marrow from mouse limbs. A cell suspension was mashed through a 70-lm filter, and neutrophils were separated from the single-cell suspension by positive selection on a MACS column using anti-Ly6G-biotin and anti-biotin microbeads (Miltenyi Biotec), as per the manufacturer's instructions.

Gou X., Yuan J., Wang H., Wang X., Xiao J., Chen J., Liu S., Yin Y, & Zhang X. (2020). IL-6 During Influenza-Streptococcus pneumoniae Co-Infected Pneumonia—A Protector. Frontiers in Immunology, 10, 3102.

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Generation of Mouse Neutrophils from BM

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Monocyte Tracking in Mouse Models

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mPEG-DSPE and NHS-PEG-DSPE were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Mouse CCR2 antibody (Clone # 475301) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA) and had a molecular weight of 593.66 Da. Both 1,1-dioctadecyl-3,3,3,3,-tetram-ethylindodicarbocyanide (DiD) and DAPI were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The CCR2 antagonist BMS CCR2 22 was purchased from Tocris Bioscience (Bristol, UK). Anti-NK1.1-biotin, anti-CD49b-biotin, anti-Ly6G-biotin, anti-MHC II-biotin, anti-CD90.2-biotin, anti-CD45R (B220)-biotin, anti-F4/80-APC, anti-CD11c-PE, and anti-Ly6C-fluorescein isothiocyanate were purchased from Miltenyi Biotec (Auburn, CA, USA). Anti-CD11b-BV421 and V500 streptavidin beads were purchased from BD Biosciences (San Jose, CA, USA), col-lagenase II was purchased from Worthington (Lakewood, NJ, USA), and DNase I was from Thermo Fisher Scientific (Waltham, MA, USA). The RAW 264.7 cells were purchased from American Type Culture Collection. All other chemicals and solvents were purchased from Thermo Fisher Scientific unless otherwise stated.

Wang J., Seo M.J., Deci M.B., Weil B.R., Canty J.M, & Nguyen J. (2018). Effect of CCR2 inhibitor-loaded lipid micelles on inflammatory cell migration and cardiac function after myocardial infarction. International Journal of Nanomedicine, 13, 6441-6451.

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Isolation of Thioglycollate-Induced Murine Neutrophils

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7–10 week old LMC or M-TRAF3^−/− mice were injected i.p. with 3 ml of 4% thioglycollate (BD), and cells were harvested by peritoneal lavage at 18 h post injection. Neutrophils were purified from peritoneal cells using anti-Ly-6G-Biotin and anti-Biotin magnetic beads following the manufacturer’s protocol (Miltenyi). Purified neutrophils were resuspended in RPMI-1640 medium containing 5% FCS, and aliquoted for stimulation.

Lalani A.I., Moore C.R., Luo C., Kreider B.Z., Liu Y., Morse HC I.I.I, & Xie P. (2014). Myeloid cell TRAF3 regulates immune responses and inhibits inflammation and tumor development in mice. Journal of immunology (Baltimore, Md. : 1950), 194(1), 334-348.

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