Anti cd62l apc cy7

Manufactured by BioLegend

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Anti-CD62L-APC-Cy7 is a fluorochrome-conjugated monoclonal antibody that targets the CD62L cell surface antigen. CD62L, also known as L-selectin, is a cell adhesion molecule expressed on various immune cells. The APC-Cy7 conjugate provides a bright fluorescent signal for flow cytometry applications.

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10 protocols using anti cd62l apc cy7

BrdU Incorporation and Flow Cytometry Analysis

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BrdU (Sigma Aldrich) was added at 1 mg/mL in the drinking water on day 7 after allergen challenge and BrdU incorporation was examined according to the manufacturer’s instructions (BD). Single cell suspensions were prepared from the lung, mLN, and spleen as described previously^{47 (link)}. These cells were counted using a hemocytometer and stained followed by flow cytometry using LSRFortessa^TM (BD). Following antibodies with fluorescent dye were used: anti-CD4 (PE, GK1.5, BioLegend), anti-CD4 (FITC, RM4-5, BioLegend), anti-TCR DO11.10 (APC, KJ1-26, eBioscience), anti-CD27 (Pacific Blue, BioLegend), anti-CD127 (PE-Cy7, BioLegend), anti-CD62L (APC-Cy7, BioLegend). The plots were analyzed with FlowJo (Tree Star Inc., Ver. 9.7.6).

Yeon S.M., Halim L., Chandele A., Perry C.J., Kim S.H., Kim S.U., Byun Y., Yuk S.H., Kaech S.M, & Jung Y.W. (2017). IL-7 plays a critical role for the homeostasis of allergen-specific memory CD4 T cells in the lung and airways. Scientific Reports, 7, 11155.

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Comprehensive Immune Profiling by Flow Cytometry

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For in-vitro studies, the following flow cytometry panel was used in conjunction with CTV and viability dye: anti-CD3 Alexa700, anti-CD4 PE-Cy7, anti-CD8 APC, and anti-CD44 PE, all at a 1:200 dilution (Biolegend). For in-vivo and ex-vivo studies, the following flow cytometry panel was used: anti-CD3 PE-Cy7, anti-CD4 Pacific Blue, anti-CD8 APC, anti-CD44 PE, anti-CD62L APC-Cy7, and anti-CD25 Alexa 700, all at a 1:200 dilution, except anti-CD25 at a 1:50 dilution (Biolegend). Endogenous Foxp3 expression was detected either via eGFP of eYFP fluorescence, which is compatible with all panels but not when used with BODIPY-FL dye (493/503) or 2-NBDG (465/540). In those assays, Foxp3-based uptake of FA or Glucose was not performed, but all other cellular subsets described were tested. All acquisition was performed using a BD Fortessa flow cytometer (BD, MD, USA) at the Northwestern University Comprehensive Flow Cytometry core.

Muroski M.E., Miska J., Chang A.L., Zhang P., Rashidi A., Moore H., Lopez-Rosas A., Han Y, & Lesniak M.S. (2017). Fatty Acid Uptake in T Cell Subsets Using a Quantum Dot Fatty Acid Conjugate. Scientific Reports, 7, 5790.

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Multiparameter Flow Cytometry Analysis of Cellular Activation

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5x10E4 to 1x10E5 cells were labelled for 30 minutes in 18 µl to 100 µl PBS + 1% BSA containing one or combinations of the following antibodies: anti-CD16-PECy7 (Biolegend; 1:1000; 3G8), anti-CD62 L-APCCy7 (Biolegend; 1:25; Greg-56), anti-CD63-APC (Biolegend; 1:100; H5 C6), anti-CD66b-PE (Biolegend; 1:100; G10F5), DAPI (Sigma-Aldrich), anti-CD3-PacificBlue (Beckman Coulter, 1: 50, UCHT-1), anti-CD14-FITC (Miltenyi, 1:100, Tük 4), anti-ICAM-1-AlexaFluor-405 (SantaCruz, 1:100, sc-107-af405), an in-house VHH against P-selectin (1:125, B10.6 [43 (link)],), or Annexin-V (250 ug/ml, VPS diagnostics, cat. nr. A705). Neutrophil activation was assessed by measuring the increase in CD62L expression [44 (link)] and the simultaneous decrease in CD16 and increase in CD63 expression [45 (link),46 (link)]; HUVEC activation was assessed by evaluating the increase in ICAM-1 expression [47 (link)]; red blood cell activation was assessed by evaluating phosphatidylserine exposure detected using annexin-V staining [48 (link)]; platelet activation was assessed by investigating P-selectin expression [49 (link)]. Surface labelling of cells was measured on a FACSCanto (BD Biosciences) or a LSR-II (BD Biosciences) flow cytometer relative to unlabelled controls.

Driedonks T.A., Mol S., de Bruin S., Peters A.L., Zhang X., Lindenbergh M.F., Beuger B.M., van Stalborch A.M., Spaan T., de Jong E.C., van der Vries E., Margadant C., van Bruggen R., Vlaar A.P., Groot Kormelink T, & Nolte-‘T Hoen E.N. (2020). Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases. Journal of Extracellular Vesicles, 9(1), 1764213.

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Multiparametric Flow Cytometry Analysis

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Cells were washed in PBS + 10% FCS and stained with primary antibodies (anti-CD45RA-BV421, anti-CD62L-APC-Cy7, anti CDCD49f-FITC, anti-CD51-APC, anti CD493-FITC, anti-CD49b-APC, anti-CD49a-APC, all Biolegend; anti-IL17a-Alexa647 and anti-CD146/MCAM-PE, both BD Bioscience) for 30 min on ice. Intracellular staining of IL-17a was performed by implementation of the CytoFAST Fix/Perm buffer set (Biolegend) exactly according to the manufacturer’s instructions. The cells were fixed in 4% PFA (Sigma) and the stainings were assessed on a BD Canto II flow cytometer.

Zondler L., Herich S., Kotte P., Körner K., Schneider-Hohendorf T., Wiendl H., Schwab N, & Zarbock A. (2020). MCAM/CD146 Signaling via PLCγ1 Leads to Activation of β1-Integrins in Memory T-Cells Resulting in Increased Brain Infiltration. Frontiers in Immunology, 11, 599936.

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Characterization of Memory T Cell Subsets

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Peripheral blood mononuclear cells (PBMCs) were separated by density centrifugation and cryopreserved. Prior to analysis on a Beckman Coulter Gallios flow cytometer with seven color acquisition, cryopreserved PBMC samples were thawed and cultured for 24 h before use to allow cells to rest and re-express cell surface molecules. Cells were stained with Aqua Live/Dead cell exclusion dye (Invitrogen) and the following fluorochrome-conjugated antibodies: anti-CD3-allophycocyanin (APC) (Becton Dickinson), anti-CD4-V450 (Becton Dickinson), anti-CD8-APC-A700 (Beckman Coulter), anti-CD45RA-phycoerythrin-Cy7 (Becton Dickinson), anti-CD62L-APC-Cy7 (BioLegend) and anti-CCR7-peridinin chlorophyll protein-Cy5.5 (Becton Dickinson). Single labeled tubes for each antibody, isotype-matched control antibodies, fluorescence-minus-one controls, dead cell exclusion and doublet discrimination were used to ensure accurate positive cut-off values and compensation matrices and to validate cell phenotype detection sensitivity and resolution. Figure 1 shows the gating strategy used to identify the memory T cell subsets. Flow cytometry data was analyzed using Kaluza 1.2 software (Beckman Coulter).

Pender M.P., Csurhes P.A., Pfluger C.M, & Burrows S.R. (2014). Deficiency of CD8+ effector memory T cells is an early and persistent feature of multiple sclerosis. Multiple Sclerosis (Houndmills, Basingstoke, England), 20(14), 1825-1832.

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Immunophenotyping of Myla2000, Hut78 Cells and Mice

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For experiments with Myla2000 and Hut78 cell lines, cells (0.5 × 10⁶) were fixed and permeabilized with Fixation/permeabilization buffer (eBioscience, 00-5123) for 30 minutes, followed by the staining with an isotype control antibody or an anti-IRF4-eFlour660 (eBioscience, 50-9858-82) in a permeabilization buffer (eBioscience, 00-8333) for 30 minutes.
For experiments with mice, cell suspensions were made by mincing lymph nodes in PBS. Cells (0.5 × 10⁶) were stained with an anti-CD3-BrilliantViolet421 (Biolegend, 100335), anti-CD4-PercP (eBioscience, 45-0042-82), anti-CD8-APC (Biolegend, 100711), anti-CD44-PE-eFlour610 (eBioscience, 61-0441), and anti-CD62L-APC-Cy7 (Biolegend, 104428) for 20 minutes and acquired in the FACS LSR-II (BD Biosciences) and analyzed with FlowJo V9.3.2 (Tree Star).

Vieyra-Garcia P.A., Wei T., Naym D.G., Fredholm S., Fink-Puches R., Cerroni L., Odum N., O'Malley J.T., Gniadecki R, & Wolf P. (2016). STAT3/5-Dependent IL9 Overexpression Contributes to Neoplastic Cell Survival in Mycosis Fungoides. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(13), 3328-3339.

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Comprehensive Immunophenotyping of Dendritic Cells and T Cells

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For in vitro flow cytometry analysis, cells were harvested and incubated with Fixable Viability dye APC-eFluor 780 (1:1000 dilution, Ebioscience, San Diego, CA) for 20 min. For DC cultures, cells were then stained using directly conjugated antibodies against murine surface antigens, as follows: anti-CD40-PE, anti-CD80-FITC, anti-CD3-PE-Cy7, anti-CD11b-PE, anti-CD11c-APC, anti-CD86-Alexa Fluor 700, anti-CD8-BV605, and anti-MHCII-Percp-Cy5.5 (1:400 dilution, Biolegend, San Diego, CA) and analyzed with a BD LSR-Fortessa Flow Cytometer (Becton–Dickinson, Franklin Lakes, NJ). For OTI T cell analysis, flow cytometry panel was performed as described: anti-CD4-PE, anti-CD8-APC, anti-CD44-PErcp-Cy5.5, and anti-CD3 PE/CY7 (1:200, Biolegend). For ex vivo flow cytometry analysis, the directly conjugated antibodies against murine surface antigens are as follows: anti-CD3-Alexa Fluor 700 (1:50), anti-CD4-PE-Cy7 (1:100), anti-CD8-BV605 (1:100), anti-CD44-PE (1:200), and anti-CD62L-APC-Cy7 (1:100) for surface staining (Biolegend). For intracellular staining, cells were fixed/permeabilized stained using the Foxp3 staining kit (Ebioscience) using Foxp3-eFluor 450 (1:100, Ebioscience), granzyme B Alexa Fluor 647 (1:50, Biolegend) as described in the product specification sheet.

Miska J., Rashidi A., Chang A.L., Muroski M.E., Han Y., Zhang L, & Lesniak M.S. (2016). Anti-GITR therapy promotes immunity against malignant glioma in a murine model. Cancer immunology, immunotherapy : CII, 65(12), 1555-1567.

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Phenotypic Characterization of T Cells

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Phenotypic characterization of T cells was carried out after alloreactivity, CD3/CD28 crosslinking approaches and aAPC stimulation, using the following antibodies: anti-CD3-peridinin-chlorophyll (PerCp) (SK7), anti-CD8-AlexaFluor700 (AF-700) (SK1), anti-CD25- allophycocyanin(APC)/phycoerythrin (PE)/Cy7/BV421 (BC96), anti-CD45RA-PE/Cy7/BV510 (HI100), anti-CD62L-APC/Cy7 (DREG-56), anti-CD69-APC/Cy7/PE/Cy7 (FN50), anti-137-APC (4B4-1), anti-CD366-APC/Cy7 (F38-2E2), anti-CD223-fluorescein-isothiocyanate (FITC) (11C3C65), anti-CD152-PE/Cy7 (L3D10) (all BioLegend, San Diego, CA, USA) and anti-CD279-PE (EH12.1), anti-CD154-PE (TRAP1) (BD). All flow cytometry analyses were performed using the FACS Canto 6c and 10c systems (BD) and BD FACSDiva Software version 8.0.1. At least 10,000 events were acquired in the CD3⁺ or CD8⁺ gate. Gates were set based on the light-scatter properties of lymphocytes.
Multimer staining was performed to monitor the frequency of A02pp65_p-positive (CMV-specific) CD8⁺ T cells. It was assessed before and after aAPC stimulation using PE or APC-conjugated HLA-A^*02:01/CMVpp65_p-specific (Immudex, Copenhagen, Denmark) dextramers. To be considered positive (multimer⁺), the sample had to (1) be a well-defined cell population and/or (2) contain ≥0.5% multimer⁺CD8⁺ T cells.

Bak S., Tischer S., Dragon A., Ravens S., Pape L., Koenecke C., Oelke M., Blasczyk R., Maecker-Kolhoff B, & Eiz-Vesper B. (2018). Selective Effects of mTOR Inhibitor Sirolimus on Naïve and CMV-Specific T Cells Extending Its Applicable Range Beyond Immunosuppression. Frontiers in Immunology, 9, 2953.

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Multicolor Flow Cytometry for Immune Cell Profiling

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PE anti-CD3, Pacific Blue anti-CD8α, biotin-conjugated anti-CD8β, PE Cy7 anti-CD4, APC Cy7 anti-CD62L, APC anti-CD103, PerCP Cy5.5 anti-TCRγδ, PE Cy7 anti-TCRβ, APC Cy7-B220, PerCP Cy5.5 anti-B220, FITC-EpCAM, PE Cy7 anti-CD11b, APC anti-F4/80, biotin-conjugated anti-IgA antibodies were purchased from BioLegend. Streptavidin-PE was purchased from BD Biosciences.

Ito K., Nakajima A., Fukushima Y., Suzuki K., Sakamoto K., Hamazaki Y., Ogasawara K., Minato N, & Hattori M. (2017). The potential role of Osteopontin in the maintenance of commensal bacteria homeostasis in the intestine. PLoS ONE, 12(3), e0173629.

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Multicolor Flow Cytometry Immunophenotyping

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Cells of interest were stained with different combinations of the following antibodies: FITC anti-CD3, brilliant violet 510 (BV510) anti-CD4, peridinin chlorophyll (PerCP)-conjugated anti-CD4, APC anti-CD8, APC/Cy7 anti-CD8, APC/Cy7 anti-CD45, PE anti-CD45, PE anti-CD45RO, APC/Cy7 anti-CD62L (all BioLegend) and PE-conjugated HLA-A*02:01/CMVpp65p-specific dextramer (Immudex, Copenhagen, Denmark). Surface staining was performed at room temperature for 20 min in the dark and washed with PBS (Lonza, Vervies, Belgium) with 0.1% human AB serum (c.c. pro). 7-amino-actinomycin D (7-AAD; BD Biosciences) was applied prior to flow cytometric analysis to exclude dead cells. All samples were analyzed by multicolor flow cytometry (FACS Canto II, FACSDiva V8.1.2 software, FlowJo_v10.7.1 software; all BD Biosciences). Gates were set based on the forward scatter versus side scatter properties of lymphocytes. At least 30,000 events were acquired in the CD3⁺ gate.

Laubert M., Bonifacius A., Dragon A.C., Mangare C., Blasczyk R., Huehn J, & Eiz-Vesper B. (2022). Enhancement of Antiviral T-Cell Responses by Vitamin C Suggests New Strategies to Improve Manufacturing of Virus-Specific T Cells for Adoptive Immunotherapy. Biology, 11(4), 536.

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