Anti cd62l pe

Manufactured by BD

Sourced in United States

Anti-CD62L-PE is a monoclonal antibody conjugated with the fluorescent dye Phycoerythrin (PE). It is used in flow cytometry applications to detect and quantify the expression of the CD62L (L-selectin) cell surface marker on various cell types.

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14 protocols using anti cd62l pe

Multiparameter Flow Cytometry of T-cell Subsets

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Purified CD8⁺ T cells, splenocytes and thymocytes were first stained for surface antigens and then treated with Foxp3 staining buffer set according to the manufacturer's directions (eBioscience). Anti-Eomes AlexaFluor 647 or eFluor 660 (Dan11mag, 1/75), anti-T-bet PE (eBio4B10, 1/100) and anti-CD49d FITC or PE (R1-2, 1/50) antibodies were purchased from eBioscience. Anti-CD8 PercP (53–6.7, 1/50), anti-CXCR3 APC (CXCR3-173, 1/50), anti-CD4 Pe-Cy7 (RM4-5, 1/100), anti-CD62L PE (1/100) or V450 (1/50) (MEL-14), anti-Bcl2 PE (3F11, 1/25), anti-Ki67 FITC (B56, 1/25), anti-CD44 FITC or V450 (IM7, 1/50), anti-CD127 Pe-Cy7 (SB/199, 1/50), anti-CD122 FITC (TM-BETA1, 1/50), anti-NK1.1 FITC (PK136, 1/50), anti-CD90.2 Pe-Cy7 (53-2.1, 1/100) and anti-IFNγ APC or PB or PE (XMG1.21/50) were purchased from BD biosciences. Anti-CD3 Pe-Cy7 (2C11, 1/100) was purchased from Biolegend.
In some experiments, brefeldin A (5 μg ml⁻¹, Sigma) was added in samples for 3 h at 37 °C before intracytoplasmic staining. Blood samples were directly stained for surface antigens and then treated with FACS lysing buffer (BD biosciences) as described in the product data sheet. All samples were fixed with 1% paraformaldehyde in PBS prior to their processing using a Cyan flow cytometer (Dako Cytomation).

Martinet V., Tonon S., Torres D., Azouz A., Nguyen M., Kohler A., Flamand V., Mao C.A., Klein W.H., Leo O, & Goriely S. (2015). Type I interferons regulate eomesodermin expression and the development of unconventional memory CD8+ T cells. Nature Communications, 6, 7089.

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Multiparametric Flow Cytometry Assay

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5-Azacytidine was obtained from Sigma-Aldrich (Munich, Germany) and used at a final concentration of 5 μM or 20 μM. All flow cytometry experiments were performed using a FACS Canto II (BD Bioscience, Heidelberg, Germany). For flow cytometry the following antibodies were used: anti-CD3-PE, anti-CD4-FITC, anti-CD8-APC, anti-CD25-PE, anti-CD45-FITC, anti-CD45RO-PE, anti-CD45RA-FITC, anti-CD62L-PE, anti-CCR7-PE, anti-HLA-DR-APC, anti-Granzyme-PE antibodies (obtained from BD Bioscience) anti-CD127-PC5, anti-IFNγ-PE/Cy7, anti-IL17-APC, anti-IL4-FITC, and anti-FoxP3-APC antibodies obtained from eBioscience (San Diego, USA).

Stübig T., Badbaran A., Luetkens T., Hildebrandt Y., Atanackovic D., Binder T.M., Fehse B, & Kröger N. (2014). 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity. Mediators of Inflammation, 2014, 418292.

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CD4+ Naïve T Cell Culture Protocol

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RPMI1640 medium containing 10% FCS was used for CD4 naïve T cell cultures. The following antibodies were purchased from BD BioSciences (San Jose, CA): anti-mouse CD3 (145-2C11), anti-mouse CD28 (37.51), anti-mouse IL-2, anti-CD4-PerCP, anti-CD4-PECy7, anti-CD8-FITC, anti-CD25-APC, anti-CD25-PE, anti-CD44-FITC, anti-CD62L-PE, anti-BrdU-FITC. Anti-FoxP3-APC, anti-IFNγ-APC, and anti-IL-17A-PE were from eBioscience (San Diego, CA). Anti-Helios, anti-CD122 and anti-GITR were from Biolegend (San Diego, CA). Recombinant mouse IL-2 and IL-7 were purchased from R&D Systems (Minneapolis, MN) and ovalbumin was from Sigma-Aldrich (St Louis, MO).

Liu C., Wang H.C., Yu S., Jin R., Tang H., Liu Y.F., Ge Q., Sun X.H, & Zhang Y. (2014). Id1 expression promotes T regulatory cell differentiation by facilitating TCR costimulation. Journal of immunology (Baltimore, Md. : 1950), 193(2), 663-672.

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Comprehensive Immune Cell Profiling

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Splenocytes were treated with ACK buffer for red cell lysis and washed with RPMI with 10% FBS. The spleen, heart, lymph node, and liver cells were stained with H2K^k-TEWETGQI multimer for 10 min at RT. The cell surface was stained for 30 min at 4°C. The following antibodies were used for surface staining: anti-CD3 APCcy7 (clone 145-2C11, BD), anti-CD8 PERCP or anti-CD8 PACIFIC BLUE (clone 53-6.7, BD), anti-CD11a FITC (clone 2D7, BD), anti-CD11c APCcy7 (clone HL3, BD), anti-CD44 FITC (clone IM7, BD), anti-CD62L PE (clone MEL-14, BD), anti-CXCR3 PERCP/Cy5.5 (clone 173, BioLegend), anti-CD27 FITC (clone LG3A10, BD), anti-CD4 PEcy7 (clone RM4-5, BD), anti-KLRG1 FITC (clone 2F1, eBioscience), anti-CD49d PEcy7 (clone R1-2, BD), anti-CD69 PERCP (clone H1.2F3, BD), anti-CD43 PEcy7 (1B11, BioLegend), anti-CD95 PEcy7 (clone JO2, BD), anti-CD25 FITC (clone LG3A10, BD), anti-CD127 PE (clone SB/199, BD), anti-CD122 FITC (clone TM-β1, BD), anti-CD38 PE (clone 90, BD), anti-β7 PERCP (clone FIB27, BioLegend), anti-CD31 FITC (clone MEC 13.3, BD), anti-CD272 PE (clone 8F4, eBioscience), anti-PD-1 FITC (clone J43, eBioscience), anti-CTLA-4 PE (clone UC10-4B9, eBioscience), and anti-CCR7 PE (clone 4B12, BD). At least 500,000 cells were acquired on a BD FACS Canto II flow cytometer and analyzed with FlowJo 8.7.

Ferreira C.P., Cariste L.M., Santos Virgílio F.D., Moraschi B.F., Monteiro C.B., Vieira Machado A.M., Gazzinelli R.T., Bruna-Romero O., Menin Ruiz P.L., Ribeiro D.A., Lannes-Vieira J., Lopes M.D., Rodrigues M.M, & de Vasconcelos J.R. (2017). LFA-1 Mediates Cytotoxicity and Tissue Migration of Specific CD8+ T Cells after Heterologous Prime-Boost Vaccination against Trypanosoma cruzi Infection. Frontiers in Immunology, 8, 1291.

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Comprehensive Immune Cell Profiling in Sepsis

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Mice were sacrificed and spleens were harvested at 24h after CLP. Splenocytes were stained with anti-CD3-Alexa 700 (BD), anti-CD4-PB (Biolegend, clone RM4–5), anti-CD8-PO (Invitrogen, clone MCD0830), anti-CD44-PerCP (Biolegend, clone IM7), anti-CD62L-PE (BD), anti-CD28-PE-Cy7 (Biolegend, clone E18) and anti-CD25-APC-Cy7 (BD). For detection of cell apoptosis, splenocytes were stained with a FITC Annexin V apoptosis detection kit with 7-AAD (Biolegend). Anti-Bcl-xL (54H6) and Bcl-2 (Biolegend, clone BCL/10C4) were used to detect engagement of the mitochondrial pathway of apoptosis while anti-CD95 (Biolegend, clone DX2) and anti-TNFR Type Ⅰ (Biolegend, clone 55R-286) were stained to detect expression of death receptors on T cells. Cells were intracellularly stained with anti-Ki-67 (Biolegend, clone 16A8) to assay for cell proliferation. Tregs were identified via intracellular staining for Foxp3-FITC (Ebioscience, clone FJK-16S) using the Foxp3 staining kit (Ebioscience). B cells were stained with anti-CD19-FITC (Biolegend). NK cells were stained with anti-NK1.1-PE (Ebioscience). Dendritic cells were stained with anti-CD11c-PE-Cy7 (BD). Neutrophils were stained with anti-Gr-1-Alexa 700 (Biolegend) and anti-CD11b-PerCP (Biolegend). Accucheck Counting Beads (Thermo Fisher Scientific) were added after staining to calculate the absolute number of cells per spleen.

Sun Y., Xie J., Anyalebechi J.C., Chen C.W., Sun H., Xue M., Liang Z., Morrow K.N., Coopersmith C.M, & Ford M.L. (2020). CD28 agonism improves survival in immunologically experienced septic mice via IL-10 released by Foxp3+ regulatory T cells. Journal of immunology (Baltimore, Md. : 1950), 205(12), 3358-3371.

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Expression and Phenotyping of CD19-CARs on T Cells

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Expression of the various CD19-CARs on T cells was detected using either biotinylated anti-mouse FMC63 scFv monoclonal antibody (Biowan) followed by staining with streptavidin-phycoerythrin (PE) (BD Biosciences), or with an allophycocyanin (APC)-labeled anti-mouse FMC63 scFv monoclonal antibody (Biowan). 1 × 10⁶ cells were stained for cell surface markers to analyze T cell differentiation and exhaust status. The following antibodies were used: anti-CD3-FITC (BD Biosciences), anti-human CD3-APC (BD Biosciences), anti-CCR7-BV421 (BD Biosciences), anti-CD 45RO-APC (BD Biosciences), anti-CD4-BB515, anti-CD8-BV510 (BD Biosciences), anti-CD45RA-PE- Cy7 (BD Biosciences), anti-CD62L-PE (BD Biosciences), anti-PD-1-APC (BD Biosciences), anti-TIM-3APC (BD Biosciences), and anti-LAG-3-APC (BD Biosciences). Samples were analyzed on either FACSCanto II flow cytometers (BD Biosciences) or Novocyte3110 (AECE).

Sun M., Xu P., Wang E., Zhou M., Xu T., Wang J., Wang Q., Wang B., Lu K., Wang C, & Chen B. (2021). Novel two-chain structure utilizing KIRS2/DAP12 domain improves the safety and efficacy of CAR-T cells in adults with r/r B-ALL. Molecular Therapy Oncolytics, 23, 96-106.

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Western Blot Analysis of Immune Cell Signaling

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All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Anti-mAbs used for Western blotting against extracellular signal-regulated kinase (ERK)1/2, p-ERK1/2, p38, p-p38 (Thr180/Tyr182), c-Jun N-terminal kinase (JNK), p-JNK (Thr183/Tyr185), IκB-α, p-IκB-α (Ser32), p-Ikkα/β (Ser176/180), and p-Akt (Ser473) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-Gα16 mAb was from Abcam (Cambridge, UK). Anti-F(ab′)2 fragment goat anti-mouse (GAM) IgG (H + L) was from Jackson ImmunoResearch (West Grove, PA, USA). Anti-CD11b-PE, anti-CD62L-PE, anti-CD81-PE, anti-CD9-APC, and anti-CD4-FITC for flow cytometry and anti-phosphotyrosine, anti-β-actin mAb for Western blotting were purchased from BD Biosciences. The mAbs used for cell stimulation were 2A1 (EMR2-specific mAb) (AbD Serotec) and mouse monoclonal IgG1 (Clone 11711) (R&D System) as described previously (18 (link)).

I K.Y., Huang Y.S., Hu C.H., Tseng W.Y., Cheng C.H., Stacey M., Gordon S., Chang G.W, & Lin H.H. (2017). Activation of Adhesion GPCR EMR2/ADGRE2 Induces Macrophage Differentiation and Inflammatory Responses via Gα16/Akt/MAPK/NF-κB Signaling Pathways. Frontiers in Immunology, 8, 373.

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Cell Surface Marker Analysis

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To analyze cell surface expression levels of the various markers, the cell suspensions (1 × 10⁶) were incubated with following mAbs: anti-CD3-APC, anti-B220-FITC, anti-CD4-PE, anti-CD4-FITC, anti-CD8-FITC, anti-NK1.1-PerCP.Cy5.5, anti-PLZF-PE, anti-CD44-FITC, anti-CD69-APC, anti-CD62L-PE, anti-CD62L-PErCP, anti-CD25-APC, anti-CD122-PE, and anti-CD127-PE (all BD Pharmingen) acquired in a FACScan (Becton Dickinson, Mountain View, CA, USA) and analyzed using FlowJo software.

Pachulec E., Neitzke-Montinelli V, & Viola J.P. (2016). NFAT2 Regulates Generation of Innate-Like CD8+ T Lymphocytes and CD8+ T Lymphocytes Responses. Frontiers in Immunology, 7, 411.

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Tonsil-Derived T-Cell Isolation and Infection

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Tonsils were obtained as de-identified specimens from discarded pathologic specimens of children without known HIV-1 infection undergoing elective tonsillectomies at Children’s Hospital Denver in accordance with the Colorado Multiple Institutional Review Board (CMIRB), which reviewed this protocol on September 23, 2008 and determined that it does not constitute human subjects research. For this reason, informed consent is not required.
Tonsils were mechanically disaggregated in sterile phosphate buffered saline. The cell suspension was filtered through a 70 micron filter (Fisher Scientific, Denver, CO) and washed with PBS. Single cell suspensions were infected with NLENG1-IRES as described [133] (link), then IL-4 (25 ng/ml) was added to the cultures and again on day 3 post infection. For flow cytometry, cells were stained with anti-CD3-PerCP, anti-CD8-PerCP-Cy5, anti-CD45RA-PE-Cy5, anti-CD62L-PE, (all from BD Pharmingen) and analyzed on a Becton Dickinson (BD) LSR II flow cytometer (UC Denver). Gating for GFP expression was on CD3+CD8^–CD45RA+ cells.

Trinité B., Chan C.N., Lee C.S., Mahajan S., Luo Y., Muesing M.A., Folkvord J.M., Pham M., Connick E, & Levy D.N. (2014). Suppression of Foxo1 Activity and Down-Modulation of CD62L (L-Selectin) in HIV-1 Infected Resting CD4 T Cells. PLoS ONE, 9(10), e110719.

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Cell Viability and Immune Cell Profiling

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Cell survival of MEFs was quantitated using the CellTiter-Glo Luminescent Cell Viability Assay per manufacturer’s instructions (Promega). The following antibodies were purchased (BD bioscience) and used for FACS studies: anti-CD4-PEcy7, anti-CD4-APC, anti-CD8-PEcy7, anti-CD8-APC, anti-CD11b–PEcy7, anti-Gr1-APC, anti-CD45.1-APC, anti-CD45.1-FITC, anti-CD45.2-APC, anti-CD45.2-FITC, anti-CD44-FITC, anti-CD62L–PE, anti-CD62L–APC, anti-TCRβ-FITC. Live cells were quantitated by flow cytometry of DAPI-negative cells. For intracellular cytokine expression, cells were incubated with PMA (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml, Sigma-Aldrich) and GolgiPlug (1 µl/ml, BD Bioscience) for 4 hrs before harvesting. Cells were stained using anti-CD4-e450 (Tonbo), anti-IFNγ−PE (BD Bioscience), anti-IL17A–Alexa647 (BD Bioscience), Live/Dead cell stain kit (Invitrogen) and Cytofix/Cytoperm kit (BD Bioscience).

Onizawa M., Oshima S., Schulze-Topphoff U., Oses-Prieto J.A., Lu T., Tavares R., Prodhomme T., Duong B., Whang M.I., Advincula R., Agelidis A., Barrera J., Wu H., Burlingame A., Malynn B.A., Zamvil S.S, & Ma A. (2015). The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis. Nature immunology, 16(6), 618-627.

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