Cd62l apc

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD62L-APC is a fluorescently labeled monoclonal antibody that binds to the CD62L (L-selectin) cell surface antigen. CD62L is expressed on various cell types, including lymphocytes, and is involved in cell-cell adhesion and trafficking. The APC (Allophycocyanin) fluorophore is conjugated to the antibody, allowing for detection and analysis of CD62L-expressing cells using flow cytometry.

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9 protocols using cd62l apc

Murine Inflammatory Marker Antibodies

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Rabbit monoclonal antibodies to murine IL-1β (clone D3H1Z), neutrophil elastase (clone E8U3X), PTP-PEST (clone D4W7W), and rabbit polyclonal antibody to SHIP1 (D1163) were from Cell Signaling Technology, rabbit polyclonal antibody to GAPDH (#G9545) from Sigma-Aldrich. The monoclonal antibodies to phosphotyrosine (clone 4G10) and PSTPIP2 (clones PSTPIP2-01 and PSTPIP2-03 (14 (link))) were produced in-house with the use of respective hybridomas. Flow cytometry antibodies Ly6G-FITC (catalog # 127606, also used for Western blot), Ly6C-PE-Cy7 (# 128018), CD11b-PE (# 101208) were from Biolegend and CD62L-APC (# 177-0621-81) was from eBioscience (ThermoFisher).

Pavliuchenko N., Duric I., Kralova J., Fabisik M., Spoutil F., Prochazka J., Kasparek P., Pokorna J., Skopcova T., Sedlacek R, & Brdicka T. (2022). Molecular interactions of adaptor protein PSTPIP2 control neutrophil-mediated responses leading to autoinflammation. Frontiers in Immunology, 13, 1035226.

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Disulfiram and Copper Gluconate Protocol

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Disulfiram (Selleck, S1680) and copper gluconate (CuGlu, Sangon Biotech, A503014) with over 99% purity were used for the study. The antibody to NPL4 (sc-365796) was obtained from Santa Cruz. The antibodies to EIF2S1 (ab32157), XBP1 (ab220783), and EIF2S1 (phosphor S51) (ab32157) were obtained from Abcam. The antibodies to CHOP (A0221), Ubiquitin (A18185), β-tubulin (A7074), and Lamin-B (A19970) were obtained from Abclonal. The antibodies to Zombie NIR™ APC-CY7(423106), CD45 FITC (103108)/PE (103106), CD11c PE (117308)/APC (117310), I-Ab PE-CY7 (116420), CD80 PE-CY5.5 (104712), CD86 APC (105012), CD3 APC (100236), CD8 PE (100708)/APC-CY7 (100712), CD4 PE (130310), CD44 PE-CY7(103010), CD62L APC (104412), Granzyme B PE-CY7(372213), and IFN-γ PE-CY5.5(505821) were obtained from eBioscience (San Diego, CA, USA).

Gao X., Huang H., Pan C., Mei Z., Yin S., Zhou L, & Zheng S. (2022). Disulfiram/Copper Induces Immunogenic Cell Death and Enhances CD47 Blockade in Hepatocellular Carcinoma. Cancers, 14(19), 4715.

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Flow Cytometry Analysis of Immune Cell Populations

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BMNC. PECs, lymph or splenic cells were washed in FACS buffer, blocked for 10 min in F_c block and incubated with various conjugated antibodies. To determine antigen specific T cells: CD3-FITC, OTI-CD8-dsRed, MHC I-Ova dextramer-APC (Immudex, Denmark and Tetramer core facility NIH, Bethesda, MD); to determine T cell activation: CD3-PerCp (145-2C11), CD4-PECy7(RM4-5), CD8-FITC (53-6.7), CD44-PE (IM-7), CD62L-APC (MEL-14); to determine monocyte populations: CD11c-APCCy7 (N418), CD11b-FITC (M1/70), Ly6C-PE (AL-21), Ly6G-APC (RB6-8C5), MHCII-PECy7 (M5/114.15.2), and CD45-PerCp (30F11), (eBioscience, San Diego, CA). Cells were analyzed using a BD FACSCanto II flowcytometer (BD Biosciences, San Jose, CA) and FlowJo analysis software v.8.7.3 (Treestar Software, Ashland, OR). Cell populations were determined by gating on CD4⁺, CD8⁺, (T cells) CD45^hi/CD11b⁺ (macrophages) and CD45^int/CD11b⁺ (microglia) Ly6CintLy6G⁺, (neutrophils) Ly6C⁺Ly6G⁻, (inflammatory monocytes) from a live cell gate (gating strategy as depicted in Supplementary Figs. 5, 7).

McGovern K.E., Nance J.P., David C.N., Harrison R.E., Noor S., Worth D., Landrith T.A., Obenaus A., Carson M.J., Morikis D, & Wilson E.H. (2021). SPARC coordinates extracellular matrix remodeling and efficient recruitment to and migration of antigen-specific T cells in the brain following infection. Scientific Reports, 11, 4549.

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Immune Cell Analysis Protocol

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The immune cells were analyzed following the standard protocol. Briefly, tumors were collected and incubated in dissociation buffer with 1640 medium (contained collagenase, hyaluronidase, and deoxyribonuclease I) at 37 °C for digest tumor tissues to the single‐cell suspension. And then the cells were stained with surface antibodies: CD3‐PE (eBioscience, Catalog: CD0304), CD4‐FITC (eBioscience, Catalog: 11‐0041‐82), CD8‐PerCP‐Cy5.5 (eBioscience, Catalog: 45‐0081‐82), CD86‐PE (eBioscience, Catalog: 12‐0862‐81), CD11b‐FITC (eBioscience, Catalog: 11‐0112‐81), F4/80‐PE‐Cy5 (eBioscience, Catalog: 15‐4801‐82), CD206‐PE (eBioscience, Catalog: 12‐2061‐82), CD44‐PE (eBioscience, Catalog: 12‐0441‐81), or CD62L‐APC (eBioscience, Catalog: 17‐0621‐81) respectively for 30 min. After fixing and perforating the cells, the intracellular markers: FOXP3‐PE (eBioscience, Catalog: 12‐5773‐82) or IFN‐γ‐PE (eBioscience, Catalog: 12‐7311‐82) were stained. The stained cells were detected using flow cytometry (Beckman Cytoflex S flow cytometer). The data were analyzed using FlowJo 10.0.

Sang W., Xie L., Wang G., Li J., Zhang Z., Li B., Guo S., Deng C, & Dai Y. (2020). Oxygen‐Enriched Metal‐Phenolic X‐Ray Nanoprocessor for Cancer Radio‐Radiodynamic Therapy in Combination with Checkpoint Blockade Immunotherapy. Advanced Science, 8(4), 2003338.

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Phenotypic Analysis of Murine Splenocytes

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For phenotypic analysis, animals were sacrificed at 24hrs utilizing CO₂ euthanasia and their spleens were harvested. The spleens were processed into single-cell suspensions and the number of cells per mL of suspension was obtained via a Nexcelom Auto Cellometer. 2x10⁶ cells were plated into a 96-well plate and stained for extracellular markers CD4—Pacific Blue (BD Biosciences, clone RM4-5), CD8—Pacific Orange (Invitrogen, clone MCD0830), CD3—APCCy7 (BioLegend, clone 17A2), CD44—PerCP (BioLegend, IM7), CD62L—APC (eBioscience, clone MEL-14), CD43—FITC (BioLegend, clone 1B11), and CD43—PE (BD Pharmingen, S7). TruCount Beads from BD Pharmingen were prepared according to the manufacturer’s instructions and used to determine absolute cell counts.

Fay K.T., Chihade D.B., Chen C.W., Klingensmith N.J., Lyons J.D., Ramonell K., Liang Z., Coopersmith C.M, & Ford M.L. (2018). Increased mortality in CD43-deficient mice during sepsis. PLoS ONE, 13(9), e0202656.

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Characterization of Aged Neutrophils

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Peripheral blood was lysed with ACK lysing buffer (Gibco, Cat# A1049201) and the nucleated cells were stained with the following conjugated monoclonal antibodies: CD62L-APC, CD115-PE-Cy7, CXCR4-PE, and Gr-1-FITC (all from eBioscience, USA). Flow cytometry was carried out on the Moflo XDP Sorter (BD Biosciences). Dead cells were excluded by FSC, SSC and Propidium iodide. Neutrophils were gated by Gr-1^hi CD115^lo SSC^hi and aged neutrophils gated by CD62L^lo CXCR4^hi within the neutrophil population.

Dutta D., Methe B., Amar S., Morris A, & Lim S.H. (2019). Intestinal injury and gut permeability in sickle cell disease. Journal of Translational Medicine, 17, 183.

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Flow Cytometry Analysis of Cell Surface Markers

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Flow cytometry was performed on FACS Canto2 and FACS Calibur instruments (Becton Dickinson, Heidelberg, Germany). Cells were stained for 30 min on ice with CD62L-APC (eBioscience) or CD27-PerCP-Cy5.5 (Biolegend, Koblenz, Germany) and washed once before analysis. For live/dead cell discrimination propidium iodide (PI) (Molecular Probes/Thermo Fisher Scientific) was added immediately before analysis. Analysis was performed using the FlowJo Software package (FlowJo, LLC, version 9.9.4). Flow cytometric monitoring of FRET signals was performed by generating the ratio of the 405-nm laser signals in the 450/50 and 510/50 nm channels using FlowJo.

Johnsen B., Kaschubowski K.E., Nader S., Schneider E., Nicola J.A., Fliegert R., Wolf I.M., Guse A.H., Nikolaev V.O., Koch-Nolte F, & Haag F. (2019). P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP. Purinergic Signalling, 15(2), 155-166.

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Phenotypic Characterization of Autoimmune T Cells

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Surface-antigen staining was performed according to standard protocols using the following antibodies: CD3-PerCpCy5.5 (BioLegend), CD4-PB (BioLegend), CD8-APC-Cy7 (eBioscience), CD25-PE (BD Bioscience), CD44-PeCy7 (BioLegend), CD62L-APC (eBioscience), anti-TCR-Vβ antibodies (BD Bioscience). For intracellular cytokine staining, 1 × 10⁶ lymphocytes were re-stimulated with 50 μg/ml myelin oligodendrocyte-derived glycoprotein (MOG)_33–55 peptide in the presence of Golgi Plug (BD Bioscience) for 6 h. The staining was performed with Cytofix/Cytoperm reagents (BD Bioscience) according to the manufacturer’s protocol. The following antibodies were used: IL-17-APC (BD Biosciences), GM-CSF-APC (BD Biosciences), IFNγ-APC (BD Biosciences). Intracellular transcription factor staining was performed using a FoxP3 staining buffer set (eBioscience) and a FoxP3-APC antibody (eBioscience) according to the manufacturer’s protocol. Flow cytometry was performed on a FACSCanto (BD Bioscience) with FACS Diva software (BD Bioscience) and analyzed with FlowJo 9.6 (Treestar).

Meister M., Papatriantafyllou M., Nordström V., Kumar V., Ludwig J., Lui K.O., Boyd A.S., Popovic Z.V., Fleming T.H., Moldenhauer G., Nawroth P.P., Gröne H.J., Waldmann H., Oelert T, & Arnold B. (2015). Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses. Frontiers in Immunology, 6, 78.

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Characterizing CD8+ T Cell Activation

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CD8+ cells were isolated from the spleens of 8–12 week old C57BL/6J mice (The Jackson Laboratory, 000664) fed standard chow maintained under IACUC approved protocols. CD8+ cells isolated to greater than 95% purity by CD8+ negative isolation kit (Miltenyi, #130-095-236). CD8+ cells were stained using the proliferative dye Cell Trace Violet (LifeTech, #C34557) before stimulation as per manufacturer’s protocol. Cells were stimulated on 5 μg/mL anti-CD3/CD28-coated plates (ebioscience, aCD3 #16-0031-85; aCD28 #16-0281-85) in RPMI 1640 media containing IL-2 (‘activated’, eBioscience, #14-8021-64, 10 ng/mL), or in tissue-culture treated plates in media containing IL-7 (‘naive’, Peprotech, #217-17), 1 ng/mL) for up to 7 days. Drug was added at the beginning of activation. Cells were removed at each day described for viability (Propidium Iodide, Sigma-Aldritch, #P4864), cell counts, proliferation, and/or surface markers of activation. Cell surface markers used were CD8-APC (ebioscience, #17-0081-82), CD62L-APC (ebioscience, #17-0621-82), CD44-PE (ebioscience, #12-0441-82), and CD62L-FITC (ebioscience, #11-0621-81). At day 4 and 7, cells were removed for intracellular granzyme B PE stain (ebioscience, #12-8898-80). All data were acquired in triplicate on a MacsQuant Analyzer (Miltenyi Biotec) and analyzed using FlowJo V10 (TreeStar software).

Liberti M.V., Dai Z., Wardell S.E., Baccile J.A., Liu X., Gao X., Baldi R., Mehrmohamadi M., Johnson M.O., Madhukar N.S., Shestov A.A., Christine Chio I.I., Elemento O., Rathmell J.C., Schroeder F.C., McDonnell D.P, & Locasale J.W. (2017). Metabolic Control Analysis Uncovers a Predictive Model for Selective Targeting of the Warburg Effect through GAPDH Inhibition with a Natural Product. Cell metabolism, 26(4), 648-659.e8.

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