Anti ly6g microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

Anti-Ly6G microbeads are magnetic beads coated with antibodies specific to the Ly6G antigen. They are designed for the isolation and enrichment of Ly6G-positive cells, such as neutrophils, from various biological samples.

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Lab products found in correlation

Automacs pro separator, by Miltenyi Biotec (2 mentions) Gm csf, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Lumaplate, by PerkinElmer (1 mentions) Na251cro4, by PerkinElmer (1 mentions) C57bl 6j mice, by Charles River Laboratories (1 mentions) Recombinant mouse c5a, by ProSpec (1 mentions) Sb225002, by MedChemExpress (1 mentions) Image pro plus, by Media Cybernetics (1 mentions) Flt3l, by Thermo Fisher Scientific (1 mentions) Hyaluronidase, by Merck Group (1 mentions) Pe anti mouse ly6g, by BioLegend (1 mentions) Mouse recombinant cxcl1, by BioLegend (1 mentions) Anti citrullinated h3, by Abcam (1 mentions) Neutrophils, by Miltenyi Biotec (1 mentions) Dnase 1, by Merck Group (1 mentions) Collagenase, by Worthington (1 mentions) Mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions) Facs aria, by Miltenyi Biotec (1 mentions) Facsaria, by BD (1 mentions)

19 protocols using anti ly6g microbeads

Murine Neutrophil-Mediated Cytotoxicity Assay

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Mice were injected subcutaneously with 20 µg recombinant human PEGylated G-CSF and after 4 days blood was collected in lithium-heparin tubes under terminal anesthesia. Murine neutrophils were isolated from blood by performing erythrocyte lysis (Biolegend) twice for 4 min at 4 °C, followed by magnetic separation using anti-Ly-6G Microbeads (Miltenyi) according to manufacturer’s instructions. Neutrophils were added in an effector-target (E:T) ratio of 40:1 and for Whole Blood (WB) assays 25 µL of whole blood was added per condition.
Target cells were labeled with radioactive chromium-51 (Na₂⁵¹CrO₄, PerkinElmer) for 2 h and washed three times. Antibodies against EGFR (A431), GD2 (IMR32) or HER-2 (SKBR3) were added in concentrations as indicated per experiment. The killing assays were incubated for 4 h at 37 °C in a humidified incubator containing 5% CO₂. Plates were centrifuged and supernatant was transferred to lumaplates (PerkinElmer) to assess radioactivity induced scintillation (in counts per minute, cpm) on a beta-gamma counter (PerkinElmer). Specific lysis was calculated using the formula: ((Experimental cpm—basal cpm)/(maximal cpm—basal cpm))*100, with maximal lysis determined by incubating target cells with 1.25% triton and minimal lysis determined by chromium release of target cells in the absence of antibodies and effector cells.

Stip M.C., Jansen J.H., Nederend M., Tsioumpekou M., Evers M., Olofsen P.A., Meyer-Wentrup F, & Leusen J.H. (2023). Characterization of human Fc alpha receptor transgenic mice: comparison of CD89 expression and antibody-dependent tumor killing between mouse strains. Cancer Immunology, Immunotherapy, 72(9), 3063-3077.

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Isolation and Stimulation of Murine Neutrophils

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C57BL/6J mice (seven‐week‐old male) were purchased from Charles River Laboratories Japan, Inc. The experiments adhered to the APS Guiding Principles in the Care and Use of Animals and were approved by the Ethical Committee for Animal Experimentation of Tokyo Medical and Dental University. Bone marrow‐derived (BM‐) neutrophils were isolated from femurs and tibias of mice using anti‐Ly‐6G MicroBeads (Miltenyi Biotec) according to manufacturer’s protocol. CD115‐positive monocytes in flow‐through fraction were used as monocyte (Fig. S1).
Isolated BM neutrophils were stimulated with 3nM recombinant mouse C5a (ProSpec‐Tany TechnoGene Ltd.) in RPMI1640 culture medium containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 0.17 mmol/L streptomycin for 4 hours. The culture supernatant containing BM neutrophils (Neut‐CS) was used in chemotaxis assay.

Dewan S.M., Osaka M., Deushi M, & Yoshida M. (2021). Complement C5a‐triggered differentiated HL‐60 stimulates migration of THP‐1 monocytic leukocytes via secretion of CCL2. FEBS Open Bio, 11(5), 1374-1381.

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Neutrophil Isolation and Transwell Migration

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Neutrophils were isolated from liver tissue by using anti-Ly6G microbeads (cat number: 130-092-332; Miltenyi Biotec, Auburn, CA). Two times 10⁵ neutrophils were placed in the upper chamber of the Transwell inserts with a pore size of 5 μm (Falcon inserts; Corning Incorporated, Corning, NY), which were loaded in wells containing medium plus 1% or 5% serum from control or α-Galcer–treated mice. In ablation experiments, anti-CXCL1 (cat number: MAB453; 500 ng/mL; R&D Systems, Minneapolis, MN) or CXCR2 antagonist SB225002 (cat number: HY-16711; 2 μmol/mL; MedChem Express, Monmouth Junction, NJ) and their corresponding control were added into the medium. Two hours later, inserts were removed and stained with crystal violet. Migrated neutrophils were counted by Image-Pro Plus; Media Cybernetics, Rockville, MD).

Chen L., Gu J., Qian Y., Li M., Qian Y., Xu M., Li J., Wen Y., Xia L., Li J., Xia Q., Kong X, & Wu H. (2019). Deletion of C-C Motif Chemokine Ligand 5 Worsens Invariant Natural Killer T-Cell–Mediated Hepatitis via Compensatory Up-regulation of CXCR2–Related Chemokine Activity. Cellular and Molecular Gastroenterology and Hepatology, 7(3), 623-639.

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Neutrophil-Mediated Tumor Cell Killing Assay

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Two days after aCD40 treatment, livers were excised and digested with 450 U/ml collagenase I, 125 U/ ml collagenase XI, 60 U/ml DNase I, 60 U/ml hyaluronidase (Sigma Aldrich), and 20 mM HEPES buffer in PBS at 37°C for 20 minutes shaking at 900 rpm, as described previously^{59 (link)}. Digested tissue was then processed through a 40 μm cell strainer, centrifuged at 1500 rpm for 5 minutes, subjected to ACK red blood cell lysis, and resuspended in PBS with 0.5% BSA. Magnetic selection with anti-Ly-6G microbeads (Miltenyi Biotec) was used to isolate neutrophils from the resulting cell suspension. Isolated neutrophils were co-cultured with MC38-H2B-GFP tumor cells (40:1 neutrophil:cancer cell ratio). GFP⁺ tumor cells were quantified 24 hours later via microscopy (DeltaView). Co-culture of cancer cells with splenocytes from untreated mice was used as a negative control condition. For DC/neutrophil co-cultures, bone marrow cells were isolated from IL-12p40 reporter (IL12p40-eYFP) mice and cultured with 300 ng/mL Flt3L (Peprotech) for 8–10 days to generate DCs. Neutrophils were co-cultured with DCs (10:1 neutrophil:DC ratio) for 24 hours, before quantifying YFP⁺ cells via microscopy (DeltaVision). Treatment with TLR7/8 agonist R848 was used as a positive control condition.

Gungabeesoon J., Gort-Freitas N.A., Kiss M., Bolli E., Messemaker M., Siwicki M., Hicham M., Bill R., Koch P., Cianciaruso C., Duval F., Pfirschke C., Mazzola M., Peters S., Homicsko K., Garris C., Weissleder R., Klein A.M, & Pittet M.J. (2023). A neutrophil response linked to tumor control in immunotherapy. Cell, 186(7), 1448-1464.e20.

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Isolation and Analysis of Neutrophil NETosis

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Circulating neutrophils were isolated from blood harvested by cardiac puncture from mice using anti–Ly-6G MicroBeads (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Bone marrow (BM)–derived leukocytes were harvested from the femurs and tibias of LDLR^−/− mice by flushing with 1.5 mmol/L ethylenediaminetetra-acetic acid in Hank’s Balanced Salt Solution. After centrifugation, 1 · 10⁶ cells were seeded on a poly-l-lysine–coated 22-mm coverslip (32 (link)). These cells were incubated in Roswell Park Memorial Institute Media 1640 with 20% each of murine plasma from wt mice fed NC or HFD, LDLR^−/− mice fed NC or HFD for 28 days or recombinant mouse CXCL1 (BioLegend, Inc., San Diego, California) for 2 h. Cells were fixed by 4% paraformaldehyde in phosphate-buffered saline (PBS[–]) and stained by anti–citrullinated H3 (Abcam, Cambridge, United Kingdom), PE-antimouse Ly-6G (BioLegend, Inc.) and 4′,6-diamidino-2-phenylindole, followed by observation with a fluorescence microscope. The obtained images were analyzed with Meta Morph. The ratio of citrullinated histone H3–positive, Ly-6G–positive neutrophils in total Ly-6G–positive neutrophils was calculated.

Osaka M., Deushi M., Aoyama J., Funakoshi T., Ishigami A, & Yoshida M. (2021). High-Fat Diet Enhances Neutrophil Adhesion in LDLR-Null Mice Via Hypercitrullination of Histone H3. JACC: Basic to Translational Science, 6(6), 507-523.

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Isolation and Characterization of Post-MI Macrophages and Fibroblasts

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Macrophages and fibroblasts were isolated from post-MI hearts by immunomagnetic separation. Hearts were enzymatically digested at 37°C for 1 hr (600U/mL collagenase, Worthington; 60U/mL DNase I, Sigma Aldrich; diluted in HBSS). Digested tissue was filtered through 30μm columns to generate single cell suspensions, then incubated with anti-Ly6G microbeads and filtered through magnetic columns to remove neutrophils (Miltenyi). Flow through was then incubated with anti-CD11b microbeads to purify macrophages, which were immediately plated in RPMI media with 0.1% fetal bovine serum (FBS) for 2 hr. For Seahorse® assays, 5 × 10⁵ cells were plated in Seahorse® 24-well cell culture plates, while 1.0–1.5 × 10⁶ cells were plated for RNA extraction and collected in 1mL Trizol. For fibroblast isolation, single cell suspensions were plated on T25 flasks for 2 hr in DMEM media with 10% FBS. Non-adherent cells were then removed and replaced with fresh DMEM + 10% FBS. Cells were allowed to grow and collected at 80–90% confluence in Trizol for RNA extraction (passage 0) or passaged with 0.25% trypsin and plated in Seahorse® 24-well cell culture plates (2 × 10⁵) overnight in DMEM + 1% FBS for Seahorse assay. For in vitro treatment, fibroblasts were exposed to 10, 25, or 50 μM DMF for 24 hr or equivalent volume of vehicle control (DMSO).

Mouton A.J., Flynn E.R., Moak S.P., Aitken N.M., Omoto A.C., Li X., da Silva A.A., Wang Z., do Carmo J.M, & Hall J.E. (2021). Dimethyl fumarate preserves left ventricular infarct integrity following myocardial infarction via modulation of cardiac macrophage and fibroblast oxidative metabolism. Journal of molecular and cellular cardiology, 158, 38-48.

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MDSC Isolation from Bone Marrow and Tumor

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Single-cell suspensions were prepared from bone marrow, spleen, and tumor followed by red blood cell removal using ammonium chloride lysis buffer. Tumor tissues were processed using Mouse Tumor Dissociation Kit according to the manufacturer’s recommendation (Miltenyi). Tumor M-MDSCs (CD45+CD11b+Ly6G−Ly6Chigh) were sorted using FACS Aria (BD Biosciences). PMN-MDSCs were purified using anti-Ly6G microbeads (Miltenyi Biotec) according to the manufacturer’s instruction or sorted using FACS Aria (CD45+CD11b+Ly6G+Ly6Clo).

Kim R., Hashimoto A., Markosyan N., Tyurin V.A., Tyurina Y.Y., Kar G., Fu S., Sehgal M., Garcia-Gerique L., Kossenkov A., Gebregziabher B.A., Tobias J.W., Hicks K., Halpin R.A., Cvetesic N., Deng H., Donthireddy L., Greenberg A., Nam B., Vonderheide R.H., Nefedova Y., Kagan V.E, & Gabrilovich D.I. (2022). Ferroptosis of tumor neutrophils causes immune suppression in cancer. Nature, 612(7939), 338-346.

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Neutrophil and Macrophage-Mediated Bacterial Killing

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Neutrophils were isolated from the peritoneum 24 h after thioglycollate i.p. injection and using anti-Ly-6G microbeads from Miltenyi Biotec Inc. (San Diego, CA). Alveolar macrophages (AM) were obtained by bronchoalveolar lavage. One hundred thousand neutrophils or AM were seeded in a flat-bottom 96-well plate and infected with K. pneumoniae (MOI 1:10). After 30 min, media was washed and cells were lysed with 1% Triton-X at different time points: 0, 15, 30, 60 and 90 min (only 0 and 30 min for AM). Results were calculated as fold change in bacterial counts compared to time 0 for each mouse sample.

Tenover F.C., Mohammed M.J., Stelling J., O'Brien T, & Williams R. (2001). Ability of Laboratories To Detect Emerging Antimicrobial Resistance: Proficiency Testing and Quality Control Results from the World Health Organization's External Quality Assurance System for Antimicrobial Susceptibility Testing. Journal of Clinical Microbiology, 39(1), 241-250.

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Isolation of myeloid cell subsets

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Mouse neutrophils were isolated from the BM and spleen using MACS technique. Briefly, single cell suspensions were prepared and incubated with anti-Ly6G MicroBeads (Miltenyi Biotec) followed by selection of Ly6G⁺ cells using LS columns. The purity of isolated Ly6G⁺ cells was more than 95% as detected by flow cytometry. All Ly6G⁺ cells expressed CD11b marker. Mouse CD11b⁺Ly6G^lo/-Ly6C⁺ M-MDSC were isolated from spleens by flow sort. For isolation of tumor-associated myeloid cells, single cell suspension was prepared using Tumor Dissociation Kit (Miltenyi Biotec). CD11b⁺Ly6G⁻F4/80⁺ tumor-associated macrophages (TAM) and CD11b⁺Ly6G⁺Ly6C⁻ tumor-associated neutrophils were isolated by flow sort. Mouse hematopoietic progenitor cells (HPCs) were isolated from the BM using a Lineage depletion kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Deng H., Lin C., Garcia-Gerique L., Fu S., Cruz Z., Bonner E.E., Rosenwasser M., Rajagopal S., Sadhu M.N., Gajendran C., Zainuddin M., Gosu R., Sivanandhan D., Shelef M.A., Nam B., Vogl D.T., Gabrilovich D.I, & Nefedova Y. (2022). A novel selective inhibitor JBI-589 targets PAD4-mediated neutrophil migration to suppress tumor progression. Cancer research, 82(19), 3561-3572.

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Isolation and Cultivation of Immune Cells

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Splenic CD3⁺ T cells or NK cells were isolated from naïve mice using anti-CD90.2 magnetic beads (Miltenyi Biotec) or NK cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. Splenic CD4⁺ or CD8⁺ T cells were isolated from naïve OT-II or OT-I mice by using CD4 (L3T4) or CD8a (Ly-2) microbeads (Miltenyi Biotec), respectively. BM-derived monocytes were isolated from naïve mice using the Monocyte Isolation Kit (Miltenyi Biotec) following the manufacturer’s protocol. Neutrophils were isolated from lung tissues of naïve or tumor-bearing mice using anti-Ly6G microbeads (Miltenyi Biotech). Viability and purity of isolated cells were assessed with flow cytometry to be above 90%.
For the primary culture of lung fibroblasts, sorted CD45⁻CD31⁻CD326⁻CD140a⁺ fibroblasts were cultured in the MesenCult^™ Basal Medium (STEMCELL Technologies). The first to third passages of the primary fibroblasts were used for the ex vivo experiments.
To prepare fibroblast-derived conditioned medium (CM), ex vivo cultured lung fibroblasts or freshly sorted lung CD140a⁺ fibroblasts were cultured in RPMI-1640 medium at a density of 5×10⁵ cells/ml for 16 hours. Then the supernatant was collected after centrifuging at 1, 000g for 20 min to remove cells and debris. The freshly collected supernatant was used as CM for further experiments.

Gong Z., Li Q., Shi J., Wei J., Li P., Chang C.H., Shultz L.D, & Ren G. (2022). Lung fibroblasts facilitate pre-metastatic niche formation by remodeling the local immune microenvironment. Immunity, 55(8), 1483-1500.e9.

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