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Anti mouse ly6c apc

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Anti-mouse Ly6C-APC is a flow cytometry reagent that binds to the Ly6C antigen expressed on the surface of mouse cells. It is conjugated to the fluorescent dye APC, allowing for the detection and analysis of Ly6C-positive cells using flow cytometry.

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Lab products found in correlation

Anti mouse cd11b efluor450, by Thermo Fisher Scientific (3 mentions) Pe anti mouse ly6g, by BioLegend (2 mentions) Apc anti mouse cd8, by BioLegend (2 mentions) Foxp3 pe, by Thermo Fisher Scientific (2 mentions) Ly6g apc cy7, by BioLegend (2 mentions) Streptavidin pe cy5, by Thermo Fisher Scientific (2 mentions) Cd11b biotin, by Thermo Fisher Scientific (2 mentions) Il 17 alexa fluor 647, by Thermo Fisher Scientific (2 mentions) Pe cy7 anti mouse cd11b, by BD (1 mentions) Cd11b, by Sony (1 mentions) Sh800 cell sorter, by Sony (1 mentions) Pacific blue anti mouse cd11b, by BioLegend (1 mentions) Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Facscanto 2, by FlowJo (1 mentions) Fitc anti mouse b220, by BioLegend (1 mentions) Apc anti mouse cd206, by BioLegend (1 mentions) Fitc anti mouse cd4, by BioLegend (1 mentions) Pe anti mouse nk 1, by BioLegend (1 mentions) Collagenase type 4, by Thermo Fisher Scientific (1 mentions) Cd16 32, by BioLegend (1 mentions)

Close spelling variants of this product

Anti-Ly6C-APC Anti-mouse Ly6C Ly6C-APC Anti-Ly6C

8 protocols using anti mouse ly6c apc

1

Isolation of Spinal Cord Microglia in EAE

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Mice in the pre-immunized and acute (dpi 17) phases of EAE were euthanized, and their spinal cords were isolated and collected in ice-cold 1× Hank’s Balanced Saline Solution (HBSS). Spinal cord and brain cells were isolated by a density-gradient technique, as previously described [11 (link)]. Briefly, spinal cord and brain tissues were minced with a tissue homogenizer to obtain a single-cell suspension. Stock isotonic Percoll® (10-fold dilution in 10× HBSS without Ca2+ and Mg2+) was added to the cell suspension to form a 30% Percoll gradient. A 70% Percoll gradient was pipetted underneath the 30% Percoll cell solution and centrifuged at 800×g for 40 min. Then, the myelin layer was removed and the mononuclear cell interphase was isolated and resuspended in 1× HBSS. After blocking with an anti-mouse cluster of differentiation (CD)16/32 monoclonal antibody (Sony Biotechnology, San Jose, CA) for 10–15 min on ice, the cells were stained with phycoerythrin (PE)-Cy7 anti-mouse CD11b (BD Pharmingen™, San Jose, CA), fluorescein isothiocyanate (FITC) anti-mouse CD45 (Invitrogen, Rockford, IL), and allophycocyanin (APC) anti-mouse Ly6C (BioLegend, San Diego, CA) monoclonal antibodies. The cells were then sorted and analyzed using an SH800 Cell Sorter (Sony Corporation, Tokyo, Japan) by gating on CD11b+CD45intLy6C− for microglial cells.
Une H., Yamasaki R., Nagata S., Yamaguchi H., Nakamuta Y., Indiasari U.C., Cui Y., Shinoda K., Masaki K., Götz M, & Kira J.I. (2021). Brain gray matter astroglia-specific connexin 43 ablation attenuates spinal cord inflammatory demyelination. Journal of Neuroinflammation, 18, 126.
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2

Multiparametric Flow Cytometry of Myeloid-Derived Suppressor Cells

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Single cell suspensions were blocked with normal mouse serum (1:10) for 5 minutes at room temperature and were then stained with antibodies at 1:100 dilutions. Pacific blue anti-mouse CD11b, APC anti-mouse Ly6C, APC anti-mouse CD8, and PE anti-mouse Ly6G were purchased from BioLegend. Viability was assessed using a LIVE/DEAD fixable dead cell stain kit (Invitrogen). V405 staining was performed according to the manufacturer’s protocol and analyzed on APC+ Mo-MDSC populations. Flow analysis was performed on a FACS Canto II and data were analyzed using FlowJo software. Sorting was performed using an iCyte Reflection system.
Haverkamp J.M., Smith A.M., Weinlich R., Dillon C.P., Qualls J.E., Neale G., Koss B., Kim Y., Bronte V., Herold M.J., Green D.R., Opferman J.T, & Murray P.J. (2014). Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways. Immunity, 41(6), 947-959.
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3

Quantifying Immune Cell Populations

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Single-cell suspensions were generated by digestion with 1 mg/mL collagenase type IV (Gibco) and 1 mg/mL DNase I (SinoBiological) in PBS for 40 min at 37°C under mild shaking. The Fc receptors were blocked using CD16/32 (14-0161-82; BioLegend) for 10 min. Single-cell suspensions were then incubated with the following antibodies purchased from BioLegend: FITC antimouse CD11b (101206), PE antimouse F4/80 (123110), APC-Cy7 antimouse CD86 (105030), APC antimouse CD206 (141708), PE antimouse Ly6G (127608), APC antimouse Ly6C (128016), PE antimouse NK1.1 (108708), FITC antimouse B220 (103206), PE antimouse CD3 (100206), FITC antimouse CD4 (100406), APC antimouse CD8 (100712), and BV421 antimouse CD45 (103134). HMDMs were stained with BV421 antihuman CD68 (333828), FITC antihuman CD14 (982502), APC antihuman CD86 (374208), and PE antihuman CD206 (321106). The cells were analyzed using the FACSCanto Flow Cytometer (BD Biosciences).
Wang C., Cheng H., Yan F., Zhang H., Zhang J., Li C., Zhao M., Shi D, & Xiong H. (2023). MicroRNA-146b protects kidney injury during urinary tract infections by modulating macrophage polarization. mBio, 14(6), e02094-23.
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4

Phagocytosis of Stressed Neutrophils

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Lithium-heparinized blood of BALB/c WT and Ncf1** mice was collected. To evaluate whether platelets or complement affect phagocytosis of SNECs samples were centrifuged at 1000 rpm for 5 min to separate the plasma from the whole blood. The plasma was carefully removed and incubated with 1:1000 Prostaglandin E1 (Sigma) and 1:250 Apyrase (Sigma). Plasma was then used without further treatment or depleted of platelets via centrifugation at 400g and collection of the supernatants. To further deplete complement, platelet free plasma was heat inactivated via incubation in a water bath (57 °C, 30min). Pellets and plasma were checked for platelets via flow cytometry (FSC/SSC and CD61-PE). Prior to adding pHrodo-labelled SNECs samples were reconstituted with either plasma, platelet depleted plasma, or platelet-depleted and heat-inactivated plasma and incubate at 37 °C for 4 h. Samples were then stained with anti-mouse CD11b-eFluor450 (eBioscience) and anti-mouse Ly6C-APC (BioLegend) for 20min on ice, followed by hypotonic water lysis of erythrocytes and determination of phagocytosis indices as described before.
Hahn J., Euler M., Kilgus E., Kienhöfer D., Stoof J., Knopf J., Hahn M., Harrer T., Hultqvist M., Olofsson P., Mokhir A., Holmdahl R., Herrmann M., Schett G., Muñoz L.E, & Hoffmann M.H. (2019). NOX2 mediates quiescent handling of dead cell remnants in phagocytes. Redox Biology, 26, 101279.
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5

Characterizing Autoimmune Neuroinflammation

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Myelin oligodendrocyte glycoprotein 35–55 (MOG35–55) peptide (MEVGWYRSPFSRVVHLYRNGK) and myelin basic protein (MBP) Ac1–11 peptide (Ac-ASQKRPSQRSK) were generated by the Protein Core Laboratory of the Blood Research Institute, BloodCenter of Wisconsin. KYC was synthesized by Biomatik (Wilmington, DE). The 2.4G2 hybridoma was obtained from the American Tissue Culture Collection. Anti-mouse CD4-APC-eFluor 780 CD11b-PE, CD11b-biotin, IL-17-Alexa Fluor 647, Foxp3-PE and streptavidin-PE Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-mouse IFN-γ-PE was purchased from BD Biosciences (San Diego, CA). Anti-mouse Ly-6C-APC and Ly-6G-APC-Cy7 were purchased from Biolegend (San Diego, CA). The SMI-32 antibody, which detects nonphosphorylated neurofilament-H was purchased from Covance (Emeryville, CA). Streptavidin Alexa 405 and goat anti-mouse Alex 456 (H + L) were purchased from Life Sciences Advanced Technologies (St. Petersburg, FL). Anti-MPO heavy chain was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Anti-rabbit IgG (H + L)-HRP was purchased from Jackson ImmunoResearch (West Grove, PA). Anti-β-actin-HRP was purchased from Sigma-Aldrich (St. Louis, MO). For all experiments, the mice were age (6–8 wk) and gender matched with both sexes being utilized.
Zhang H., Ray A., Miller N.M., Hartwig D., Pritchard KA J.r, & Dittel B.N. (2015). Inhibition of Myeloperoxidase at the Peak of Experimental Autoimmune Encephalomyelitis Restores Blood-Brain-Barrier Integrity and Ameliorates Disease Severity. Journal of neurochemistry, 136(4), 826-836.
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6

Murine Model of Experimental Autoimmune Encephalomyelitis

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Myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide (MEVGWYRSPFSRVVHLYRNGK) was generated by the Protein Core laboratory of the Blood Research Institute, BloodCenter of Wisconsin. The 2.4G2 hybridoma was obtained from the American Tissue Culture Collection. Anti-mouse CD4-APC-eFluor 780, CD25-Alexa Fluor 700, CD11b-PE, CD11b-biotin, IL-17-Alexa Fluor 647, CD11c-Biotin, B220-Biotin, CD8-Biotin, CD11b-biotin, Foxp3-PE and streptavidin-PE Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-mouse B220-PE-Texas Red, IFN-γ-PE, anti-active caspase 3-FITC and anti-human Ki67-FITC were purchased from BD Biosciences (San Diego, CA). Anti-mouse Ly-6C-APC and Ly-6G-APC-Cy7 were purchased from Biolegend (San Diego, CA). Anti-mouse/rat Neuropilin-1-APC was obtained from R&D Systems (Minneapolis, MN). Monoclonal antibodies SMI-32 (anti-nonphosphorylated neurofilament-H) and SMI-99 (anti-myelin basic protein (MBP)) were purchased from Covance (Emeryville, CA). Strepavidin Alexa 405 and goat anti-mouse Alexa 546 (H + L) were purchased from Life Sciences Advanced Technologies (St. Petersburg, FL). Anti-Biotin microbeads were purchased from MiltenyiBiotec (Auburn, CA).
Ray A., Basu S., Miller N.M., Chan A.M, & Dittel B.N. (2014). An Increase in Tolerogenic Dendritic Cell and Natural T Regulatory Cell Numbers During EAE in Rras−/− Mice Results in Attenuated Disease. Journal of immunology (Baltimore, Md. : 1950), 192(11), 5109-5117.
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7

Phagocytosis of SNECs by Mouse Leukocytes

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Lithium-heparinized blood of BALB/c WT and Ncf1** mice was collected. To determine whether plasma factors affected phagocytosis of SNECs samples were centrifuged at 1000 rpm for 5 min to separate the plasma from the whole blood. Then the plasma was carefully removed and pooled Ncf1** plasma was added to WT cells and vice versa. As a control, cells were also incubated with pooled autologous plasma from the same genotype. Samples were then mixed with pHrodo-labelled SNECs and incubated at 37 °C for up to 4 h. Samples were then stained with anti-mouse CD11b-eFluor450 (eBioscience) and anti-mouse Ly6C-APC (BioLegend) for 20min on ice, followed by hypotonic water lysis of erythrocytes and analysis using a Beckman Coulter Gallios™. The PhIx was then calculated as previously described.
Hahn J., Euler M., Kilgus E., Kienhöfer D., Stoof J., Knopf J., Hahn M., Harrer T., Hultqvist M., Olofsson P., Mokhir A., Holmdahl R., Herrmann M., Schett G., Muñoz L.E, & Hoffmann M.H. (2019). NOX2 mediates quiescent handling of dead cell remnants in phagocytes. Redox Biology, 26, 101279.
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8

Phagocytosis Assay for Neutrophils

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Lithium-heparinized blood from BALB/c WT and Ncf1** mice or human NHD and SLE/CGD patients was mixed with SNECs in a 1:2 ratio and incubated at 37 °C. In some experiments, the blood was preincubated with varying concentrations of H2O2 (10 min), of the aminoferrocene compound MIS43 (10 min) [32 (link)], varying concentrations of catalase (Sigma) or butylated hydroxyanisole (BHA, Sigma) for 15 min before adding SNECs. Cells were stained with anti-mouse CD11b-eFluor450 (eBioscience), anti-mouse Ly6C-APC (BioLegend), Ly6G-PE/Cy7 (BioLegend), CCR2-FITC (BioLegend), I-A/I-E (MHC II)-Alexa Fluor 700 (BioLegend) or anti-human CD14-eFluor450 (eBioscience), CD16-APC/Cy7 and CCR2-PerCP/Cy5.5 (BioLegend), respectively. Samples were subjected to hypotonic water lysis of erythrocytes and analyzed using a Beckman Coulter Gallios™ flow cytometer. The Phagocytosis index (PhIx) was calculated in the following way: PhIx = (%PI/pHrodo-positive phagocytes x MFIPI/pHrodo). For the analysis of cytokine/chemokine production upon phagocytosis, plasma was isolated from blood incubated with SNECs (3 h) and subjected to LegendPlex bead-based assay (BioLegend).
Hahn J., Euler M., Kilgus E., Kienhöfer D., Stoof J., Knopf J., Hahn M., Harrer T., Hultqvist M., Olofsson P., Mokhir A., Holmdahl R., Herrmann M., Schett G., Muñoz L.E, & Hoffmann M.H. (2019). NOX2 mediates quiescent handling of dead cell remnants in phagocytes. Redox Biology, 26, 101279.
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