Cd62l clone mel 14

Manufactured by BioLegend

CD62L (clone MEL-14) is a lab equipment product from BioLegend that detects the expression of the L-selectin (CD62L) cell surface molecule. L-selectin is involved in the homing of lymphocytes to secondary lymphoid organs and sites of inflammation.

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16 protocols using cd62l clone mel 14

Multiparametric Flow Cytometry Immunophenotyping

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Fluorochrome-conjugated mAb directed against specific antigens and isotype-matched control antibodies were purchased from BD Pharmingen (San Diego, CA): CD62L (clone MEL-14, fluorophore BV510), BioLegend (San Diego, CA): CD11b (M1/70, BV421), CD3 (17A2, PE), CD8a (53-6.7, Alexa Fluor 488), CD4 (RM4-5/GK1.5, BV605 and BV785), CD44 (IM7, PerCP/Cy5.5), CD45 (30-F11, PE/Cy7 and PE/Dazzle™), CX3CR1 (SA011F11, BV605), Ly-6G (1A8, BV510), Ly-6C (HK1.4, PerCP/Cy5.5), CD69 (H1.2F3, BV711), CD103 (2E7, APC), PD-1 (29F.1A12, PE/Cy7), CD49a (HMα1, APC), and CD127 (A7R34, BV421).

Cassidy B.R., Zhang M., Sonntag W.E, & Drevets D.A. (2020). Neuroinvasive Listeria monocytogenes infection triggers accumulation of brain CD8+ tissue-resident memory T cells in a miR-155-dependent fashion. Journal of Neuroinflammation, 17, 259.

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Flow Cytometry Analysis of Immune Cells

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Single-cell suspensions were washed and stained with Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher, L10119) followed by staining of extracellular antigens with fluorochrome labeled antibodies. The Fixation/Permeabilization Solution Kit (BD Biosciences, 554714) was used for exposing cytoplasmic antigens. Fluorochrome-labeled antibodies against mouse antigens was used in different combinations. CD44 (clone IM7), CD45.1 (clone A20), CD45.2 (clone 104), CD8 (clone 53–6.7), CTLA-4 (clone UC10-4F10-11), were purchased from BD Biosciences. CD62L (clone MEL-14), and ICOS (clone C398.4A) were purchased from BioLegend. Anti-mouse Granzyme B (clone GB12) was purchased from Thermo Fisher. Cell counting was performed with CountBright Absolute Counting Beads (Thermo Fisher, C36950). Samples were processed in a FACSCanto II flow cytometer (BD Biosciences). Data analysis was performed with FlowJo, version 8.8.7 (Tree Star).

Rundqvist H., Veliça P., Barbieri L., Gameiro P.A., Bargiela D., Gojkovic M., Mijwel S., Reitzner S.M., Wulliman D., Ahlstedt E., Ule J., Östman A, & Johnson R.S. (2020). Cytotoxic T-cells mediate exercise-induced reductions in tumor growth. eLife, 9, e59996.

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Influenza Vaccine Formulations and Immunological Analysis

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The following influenza vaccine formulations were purchased from Moore Medical: FluMist Quadrivalent, 2014–2015, live-attenuated vaccine (MedImmune) and Fluzone Quadrivalent, 2014–2015, inactivated virus vaccine (Sanofi Pasteur) directed against A/California/7/2009 (H1N1)pdm09, A/Texas/ 50/2012 (H3N2), B/Massachusetts/2/2012, and B/Brisbane/60/2008; and 2015-2016 FluMist Quadrivalent and Fluzone Quadrivalent formulations directed against A/California/7/2009 (H1N1)pdm09, A/ Switzerland/9715293/2013 (H3N2), B/Phuket/3073/2013, and B/Brisbane/60/2008 strains. Fluorescently conjugated antibodies for Thy1.2 (clone 53-2.1, BioLegend), CD4 (clones RM4-5, BD Biosciences, and GK1.5, eBioscience), CD8 (clone 53-6.7, BioLegend), CD44 (clone IM7, BioLegend), CD62L (clone MEL- 14, BioLegend), CD69 (clone H1.2F3, eBioscience), and CD103 (clone 2E7, eBioscience) were purchased. The influenza NP–specific (NP_366–374) H-2D^b tetramer was purchased from MBL International. FTY720 was purchased from Cayman Chemical Company.

Zens K.D., Chen J.K, & Farber D.L. (2016). Vaccine-generated lung tissue–resident memory T cells provide heterosubtypic protection to influenza infection. JCI Insight, 1(10), e85832.

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Comprehensive Flow Cytometry Panel for Mouse T Cells

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Flow cytometry monoclonal antibodies against mouse antigens (CD4, clone RM4-5; CD8a, clone 53–6.7; CD62L, clone MEL-14; IFN-γ, clone XMG1.2; IL-2, clone JES6-16E3; Ifnar1, clone MAR1-5A3; PD-1, clone RMP1-30; T-bet, clone 4B10) were obtained from Biolegend. Tim-3 monoclonal antibody (clone 5D12) was a gift from V. Kuchroo. Carboxyfluorescein succinimidyl ester (CFSE) was obtained from Biolegend. The following recombinant cytokines and antibodies were used for in vitro T cell cultures: mIFN-β (PBL InterferonSource, indicated concentrations), mIL-12 (R&D Biosystems, 10 ng mL^-1), mIL-2 (Miltenyi, 5 ng mL^-1), hTGF-β (Miltenyi, 3 ng mL^-1), mIL-6 (Miltenyi, 20 ng mL^-1), mIL-23 (R&D Biosystems, 20 ng mL^-1), anti-mIL-4 (BioXCell; clone 11B11, 10 μg mL^-1), anti-CD3 (eBioscience; Functional Grade Purified, clone 145-2C11, 2 μg mL^-1), anti-CD28 (Biolegend; LEAF-purified, clone 37.51, 2 μg mL^-1). Western blotting primary antibodies were obtained from BD Transduction Laboratories (Stat1, clone 42/Stat1, dilution 1:1000; Stat1 pY701, clone 14/P-STAT1, dilution 1:1000; Stat4 pY693, clone 42/Stat1, dilution 1:500), Cell Signaling (Stat4, clone C46B10, dilution 1:500) or Millipore (GAPDH, mAb374, dilution 1:1000). Anti-mouse or-rabbit secondary antibodies were obtained from Jackson Immunoresearch and were used at 1:10000.

Boivin N., Baillargeon J., Doss P.M., Roy A.P, & Rangachari M. (2015). Interferon-β Suppresses Murine Th1 Cell Function in the Absence of Antigen-Presenting Cells. PLoS ONE, 10(4), e0124802.

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Multiparametric Flow Cytometry of Immune Cells

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At indicated time points post-infection, the spleen and mesenteric lymph nodes from male and female mice were collected and mechanically disrupted to obtain a single-cell suspension. RBC lysis buffer from BioLegend (catalog # 420301) was used to remove erythrocytes. Afterward, the cells were washed and incubated with TruStain fcX (CD16/CD32, Clone 93, BioLegend, catalog # 101320) to prevent non-specific binding, and the immune cells of interest were then stained with appropriate surface antibodies. The samples were subjected to analysis using a BD LSRFortessa flow cytometer in combination with FlowJo software (BD Biosciences). The following mouse antibodies, in various fluorochrome combinations, were utilized for the staining: CD4 (clone GK1.5, BioLegend, catalog #100412, #100406), CD8a (clone 53-6.7, BioLegend, catalog, #100707 #100711), CD11a (clone M17/4, BioLegend, catalog # 101106), CD62L (clone MEL-14, BioLegend, catalog #104438), CD49d (clone R1-2, BioLegend, catalog #103618).

Pattnaik A., Dhalech A.H., Condotta S.A., Corn C., Richer M.J., Snell L.M, & Robinson C.M. (2024). A viral-specific CD4+ T cell response protects female mice from Coxsackievirus B3 infection. Frontiers in Immunology, 14, 1327384.

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Multiparametric Flow Cytometry Analysis

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Fluorescently labeled monoclonal antibodies against the following mouse antigens were used: CD3 (clone 145–2 C11, BD Biosciences), CD4 (clone RM4-5, BioLegend), CD8a (clone 53–6.7, BioLegend), CD8b (clone YTS156.7.7, BioLegend), CD11a (clone M17/4, eBioscience), CD127 (clone A7R34, Thermo Fisher), KLRG-1 (clone 2F1, Thermo Fisher), CD44 (clone IM7, BioLegend), CD49a (clone Ha31/8, BD Biosciences), CD62L (clone MEL-14, BioLegend), CD69 (clone H1.2F3, BD Biosciences), CD38 (clone 90, Thermo Fisher) and CD103 (clone 2E7, Thermo Fisher). Cells were stained according to our previously published protocol.33 (link) 7-AAD (A1310, Invitrogen) staining was used to exclude dead cells. E7-specific CD8⁺ T cells were quantified using MHC class I tetramers for the RAHYNIVTF epitope. Flow cytometric acquisition was performed on a BD Fortessa flow cytometer (BD Biosciences).

van der Gracht E.T., Schoonderwoerd M.J., van Duikeren S., Yilmaz A.N., Behr F.M., Colston J.M., Lee L.N., Yagita H., van Gisbergen K.P., Hawinkels L.J., Koning F., Klenerman P, & Arens R. (2020). Adenoviral vaccines promote protective tissue-resident memory T cell populations against cancer. Journal for Immunotherapy of Cancer, 8(2), e001133.

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Multicolor FACS Analysis of Immune Cells

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Antibodies directed against murine CD3 (clone 145-2C11, Biolegend), CD4 (clone RM4-5, Biolegend), CD8 (clone 53-6.7, Biolegend), NK1.1 (clone PK136, Biolegend), CD62L (clone MEL-14, Biolegend), CD44 (clone IM7, Biolegend), and CD19 (clone 6D5, Biolegend) were used in multicolor FACS analysis. Samples were washed and resuspended in cold flow cytometry staining buffer (1% BSA/PBS); stained for 30 min in the dark before a final wash and acquisition. All samples were acquired on a BD Fortessa Flow cytometer running FACS DIVA software. Analysis was performed using FlowJo X software (TreeStar; OR, United States).

McCormack R.M., de Armas L.R., Shiratsuchi M., Fiorentino D.G., Olsson M.L., Lichtenheld M.G., Morales A., Lyapichev K., Gonzalez L.E., Strbo N., Sukumar N., Stojadinovic O., Plano G.V., Munson G.P., Tomic-Canic M., Kirsner R.S., Russell D.G, & Podack E.R. (2015). Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria. eLife, 4, e06508.

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Isolation and Characterization of Immune Cells from IKKβ Liver

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ELSs were dissected under binocular from IKKβ(EE)^Hep livers and digested for 30 minutes in 500µl digestion buffer (HBSS with 0.2 mg/ml collagenase IV and 0.1 mg/ml DNase1) at 37°C with gentle agitation. The cells were strained through 40µm filter by washing with cold DMEM, centrifuged for 15 minutes, RBCs were lysed for 10 minutes at 25° with erythrocytes lysis buffer, washed again and resuspended in 0.5ml DMEM and kept on ice for a few hours until staining. Viability of isolated immune cells was around 85% as determined by Trypan blue. Cells were resuspended and stained in PBS supplemented with 1% fetal calf serum and 1mM EDTA. Samples were stained and then analyzed by flow cytometry using a Gallios and Kaluza software (Beckman Coulter), or by fluorescence-activated cell sorter (FACS). Antibodies used for flow cytometry and FACS analysis: CD4 (clone RM-4.5, catalog#: 100536, BioLegend), CD8 (clone 53–6.7, catalog#: 65–0081 and 75–008, Tonbo), F4/80 (clone BM8, catalog#: 123127, BioLegend), CD11b (clone M1/70, catalog#: 101224, BioLegend), MHCII (clone KH74, catalog#: 115303, BioLegend), CD45.2 (clone 104, catalog#: 109807, BioLegend), NK1.1 (clone PK136, catalog#: 12–5941-83, eBioscience), TCRβ (clone: H57–597, catalog#: 35–5961, Tonbo), CD44 (clone IM7, catalog#: 103127, BioLegend), CD62L (clone MEL-14, catalog#: 104417, BioLegend), B220 (Catalog#: 553090, BD Pharmingen).

Finkin S., Yuan D., Stein I., Taniguchi K., Weber A., Unger K., Browning J.L., Goossens N., Nakagawa S., Gunasekaran G., Schwartz M.E., Kobayashi M., Kumada H., Berger M., Pappo O., Rajewsky K., Hoshida Y., Karin M., Heikenwalder M., Ben-Neriah Y, & Pikarsky E. (2015). Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma. Nature immunology, 16(12), 1235-1244.

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Murine T-cell Phenotyping Protocol

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Recombinant mouse IL-2 (not carrier-free) was obtained from R&D Systems. Antibodies were as follows: CD3ε (clone 17A2, eBioscience), CD4 (clone RM4-5, eBioscience), FoxP3 (clone FJK-16s, eBioscience), CD25 (clone 3C7, BioLegend), CD44 (clone IM7, BioLegend), CD45Rb (clone C363.16A, eBioscience), CD62L (clone MEL-14, BioLegend).

Jun J.C., Jones M.B., Oswald D.M., Sim E.S., Jonnalagadda A.R., Kreisman L.S, & Cobb B.A. (2017). T cell-intrinsic TLR2 stimulation promotes IL-10 expression and suppressive activity by CD45RbHi T cells. PLoS ONE, 12(7), e0180688.

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Multiparameter Immune Cell Profiling

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Cells were isolated and treated as described for the respective experiment and analysis of cell surface antigen expression was performed. For intracellular staining, cells were fixed with 1% paraformaldehyde and permeabilized with 0.1% saponin. The following fluorescent-labeled antibodies were used: CD4 (clone RM4-5), CD8 (53-6.7), CD11b (clone M1/70), CD25 (clone PC-61), CD44 (IM7), CD45 (clone 30-F11), CD62L (clone MEL-14), CD69 (clone H1.2F3), CD155 (clone 3F1), CD172a (clone P84), CD200 (clone OX-90), CD273 (clone TY25), CD274 (clone MIH5), CD71 (clone RI7217), Gr1 (clone RB6-8C5), Ly6G (clone 1A8), Ter119 (clone TER-119) (all BioLegend or BD Bioscience, Franklin Lakes, NJ). Flow cytometry acquisition and analysis were performed on an Attune Flow Cytometer (Life Technologies, Foster City, CA).

Beckmann N., Huber F., Hanschen M., St. Pierre Schneider B., Nomellini V, & Caldwell C.C. (2020). Scald Injury-Induced T Cell Dysfunction Can Be Mitigated by Gr1+ Cell Depletion and Blockage of CD47/CD172a Signaling. Frontiers in Immunology, 11, 876.

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