Cd62l pe

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD62L-PE is a fluorescently-labeled antibody that binds to the CD62L (L-selectin) surface marker on cells. It is commonly used in flow cytometry applications to identify and quantify CD62L-expressing cells.

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Close spelling variants of this product

Anti-CD62L-PE (7 citations) Anti-CD62L PE-Cy7 (11 citations) Anti-mouse CD62L PE (2 citations) CD62L-PE-Cy7 (12 citations) CD62L (58 citations)

8 protocols using cd62l pe

Isolation and Phenotyping of Immune Cells

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Mononuclear cells from liver and spleen and stromal vascular cells (SVC) from AT (epididymal and perirenal depots) were isolated as described previously (Stefanovic‐Racic et al. 2012). Cell suspensions (2 × 10⁶ cells/sample) were preincubated with anti‐CD16/32 (Fc “blocking” antibodies [Abs]) for 15 min at 4°C, then stained with either fluorescent‐labeled Abs or IgG isotype controls for 30 min at 4°C. The following Abs were used: CD8/PerCP and CD45/PerCP (BD Biosciences, San Jose, CA), CD4/FITC, CD62L/PE, NK1.1/PeCy7, B220/v450, CD3/APC, CD86/FITC, MHC2/PE, CD11b/PeCy7, B220/v450, CD11c/APC, and F4/80/Alexa780 (eBiosciences, San Diego, CA). Following incubation with Abs, liver and AT cells were incubated in Aquaporin dye (Invitrogen, Grand Island, NY) for 15 min, to distinguish live from dead cells, then fixed in 4% paraformaldehyde (Fisher, Waltham, MA) before being analyzed using a FACSCalibur flow cytometer and FACSDiva software (BD Biosciences). A proportion of up to 1% false‐positive events were accepted in the isotype control samples.

Krishna K.B., Stefanovic‐Racic M., Dedousis N., Sipula I, & O'Doherty R.M. (2016). Similar degrees of obesity induced by diet or aging cause strikingly different immunologic and metabolic outcomes. Physiological Reports, 4(6), e12708.

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Stimulation and Intracellular Cytokine Analysis of T-cell Subsets

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Freshly isolated PBMCs (Day 0) and the expanded VSTs (Day 8) were resuspended at 1.0 × 10⁶ cells per mL⁻¹ and plated in 96 well plates (200 μL per well). The cells were then stimulated with 50 ng of PHA (positive control), RPMI alone (negative control) or viral peptides for 1 hour at 37 °C and for a subsequent 4 hours at 37 °C in presence of Brefeldin A (1 μg/mL). Following incubation, PBMCs and the expanded VSTs were washed twice with PBS and surface stained for 45 minutes with CD45RA FITC, CD62L PE and CD8 PerCP (ebioscience, San Diego, CA, USA) mAbs. The cells were then washed once more in PBS and fixed overnight at 4 °C in 4% Paraformaldehyde (Sigma Aldrich, St Louis, MO, USA). Excess fixating agent was washed off twice with permeabilization buffer (PBS+0.2% saponin) and cells were incubated for 45 minutes in permeabilization buffer and an anti-TNF-α mAb conjugated to APC (ebioscience, San Diego, CA, USA). Intracellular expression of TNF-α by the different T-cell subsets with and without peptide stimulation was analyzed on a BD accuri C6 flow cytometer.

Spielmann G., Bollard C.M., Kunz H., Hanley P.J, & Simpson R.J. (2016). A single exercise bout enhances the manufacture of viral-specific T-cells from healthy donors: implications for allogeneic adoptive transfer immunotherapy. Scientific Reports, 6, 25852.

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Multicolor Flow Cytometry Panel

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Commercial antibodies (clones, fluorophores and sources) are as follows: CD3-FITC (145–2C11), CD62L-PE (Mel-14), CD4-PErCP/Cy5.5 (RM4–5), CD8-PerCP/Cy5.5 (53–6.7), CD8-APC (53–6.7), CD209b-APC (22D1), IgM-FITC (II/41), CD4-PE (GK1.5), CD25-AF488 (PC61.5), Streptavidin-PE, CD169-PE/Cy7 (3D6.112) (eBioscience, San Diego, CA); CD11b-FITC (M1/70), CD49d-FITC (R1–2), TCRβ-FITC (GL3), CD21/35-PE (7E9), CD23-APC (B3B4), Ly6G-PE (RB6–8C5), Ly6C-PerCP/Cy5.5 (HK1.4), CD11c-PE/Cy7 (N418), B220-PerCP/Cy5.5 (RA3–6B2), B220-APC (RA3–6B2), B220-APC/Cy7 (RA3–6B2), CD69-BV421 (H1.2F3),CD23-Biotin (B3B4), F4/80-APC (BM8), Ly6G-BV421 (1A8), CD45-Pacific Blue (30-F11), CD45-BV510 (30-F11), CD43-PE (1B11), CD24-PE/Cy7 (M1/69), IgD-Pacific Blue (11–26c.2a), CD69-PE/Cy7 (H1.2F3), IgD-PerCP/Cy5.5 (11–26c.2a), Ly51-AF647 (6C3), CD3-Pacific Blue (17A2), CD3-PE (17A2), TCRγ/δ-biotin (GL3), Streptavidin-PE/Cy7 (Biolegend, San Diego, CA); Siglec-F-PE (E50–2440) (BD Biosciences, San Jose, CA). Samples were preincubated with 1 μg Fc-block (2.4G2 hybridoma; ATCC).
Cells were acquired either on the BD Biosciences LSR Fortessa or with a BD FACScan flow cytometer with DxP multi-color upgrades by Cytek Development Inc. (Woodland Park, NJ) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR).

Anaya E.P., Lin X., Todd E.M., Szasz T.P, & Morley S.C. (2021). Novel mouse model reveals that serine phosphorylation of L-plastin is essential for effective splenic clearance of pneumococcus. Journal of immunology (Baltimore, Md. : 1950), 206(9), 2135-2145.

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Flow Cytometric Immunophenotyping and ATP Detection

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Cell suspensions were stained with the following antibodies: CD4-FITC (1 μg/ml), CD8-APC (0.4 μg/ml), CD62L-PE (0.4 μg/ml), CD69-PE (0.4 μg/ml) (all from e-Bioscience, Frankfurt, Germany), and CD49e-PE (0.4 μg/ml) (Biolegend, Fell, Germany) and analyzed by flow cytometry. For detection of ATP, draining LNs were removed from sensitized mice, disrupted with tweezers, and assayed for ATP (ATPlite, Perkin Elmer). For DC–T-cell co-cultures, DCs were prepared as previously described (Ring et al., 2010b (link)). Co-cultures of 1 × 10⁶ T cells and 1 × 10⁵ DCs or 5 × 10⁴ DCs were set up in 1 ml complete medium, and tissue culture supernatant was assessed for IL-10 and IFN-γ by ELISA (Ready-SET-Go, eBioscience, Frankfurt, Germany).

Mahnke K., Useliene J., Ring S., Kage P., Jendrossek V., Robson S.C., Bylaite-Bucinskiene M., Steinbrink K, & Enk A.H. (2016). Down-Regulation of CD62L Shedding in T Cells by CD39+ Regulatory T Cells Leads to Defective Sensitization in Contact Hypersensitivity Reactions. The Journal of investigative dermatology, 137(1), 106-114.

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Phenotyping T-cell Subsets by Flow Cytometry

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Freshly isolated PBMCs and the expanded VSTs (1.0 × 10⁶) were labeled with pre-diluted monoclonal antibodies (mAbs) in a 4-color direct immunofluorescence assay. Cells were incubated at room temperature in the dark for 45-minutes. The mAb combinations consisted of CD45RA FITC, CD62L PE, CD4 PerCP or CD8 PerCP and CD3 APC (ebioscience, San Diego, CA, USA). This allowed for the determination of naïve (NA; CD45RA+/CD62L+), central memory (CM; CD45RA−/CD62L+), effector memory (EM; CD45RA−/CD62L−) and CD45RA+ EM (EMRA; CD45RA+/CD62L−) subsets within the CD4+ and CD8+ T-cells29 (link)30 (link) before and after expansion. The phenotypes of the freshly isolated PBMCs (Day 0) and the expanded VSTs (Day 8) were assessed on a BD Accuri C6 flow cytometer (Accuri, Ann Arbor, MI, USA) as previously described22 (link). The total number of T-cell subsets in blood before and after exercise was determined by multiplying the percentage of cells staining positive for the appropriate surface markers in the flow cytometry lymphocyte gate by the total blood lymphocyte count (Table 2).

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Naïve CD4+ T Cell Transfer Colitis

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Naïve CD4⁺ T cell were isolated from C57BL/6 (CD45.1) mouse spleens and lymph nodes with a mouse CD4⁺ T cell isolation kit as per the manufacturer's instructions (Stemcell Technology, Canada). The isolated CD4⁺ T population was labeled with CD45Rb-FITC, CD62L-PE, and CD4-APC (eBiosciences, San Diego, CA). CD4⁺CD62L⁺CD45RB^hi T cells were sorted by flow cytometry. Then 3.5 × 10⁵ cells were injected into RAG1^−/− mice by intraperitoneal (IP) injection. Mouse body condition, the form of their stool and weight were monitored at least weekly. Mice started to lose weight and develop looser stools around 3–4 weeks in our facility indicating that colitis was present.

Chen X., Berin M.C., Gillespie V.L., Sampson H.A, & Dunkin D. (2021). Treatment of Intestinal Inflammation With Epicutaneous Immunotherapy Requires TGF-β and IL-10 but Not Foxp3+ Tregs. Frontiers in Immunology, 12, 637630.

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Lymphocyte Immunophenotyping by Flow Cytometry

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Lymphocyte stainings were performed by incubation for 20 min on ice using conjugated the following mAbs (eBioscience, San Diego, CA, USA): CD8-FITC (#11-0081-82), CD4-FITC(#MCD0401), CXCR3-APC (#17-1831-82), CCR5-PE (#12-1951-81), CD62L-PE (#12-0621-81), CD44-APC (#17-0441-81), CD25-APC (#17-0251-82), B220 FITC (#11-0452-82), CD5-PE (#12-0051-82), CD23-APC (#MCD2305).

Baur J., Otto C., Steger U., Klein-Hessling S., Muhammad K., Pusch T., Murti K., Wismer R., Germer C.T., Klein I., Müller N., Serfling E, & Avots A. (2018). The Transcription Factor NFATc1 Supports the Rejection of Heterotopic Heart Allografts. Frontiers in Immunology, 9, 1338.

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Neutrophil Surface Marker Analysis

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Surface activation markers and cell viability markers in blood derived human neutrophils were analyzed using Flow Cytometry analyzer of either the BD LSR II or the BD LSR Fortessa series (Becton Dickinson) and analyzed with FlowJo software (Tree Star). The following antibodies were used: Anti-human: CD15-APC, CD62L-PErCP-efluor710, CD66b-PE-Cy7. Anti-mouse: Ly6G(1A8)-APC-Cy7, CD62L-PE, CD11b-PE-Cy7 (all from e-Bioscience). Cell viability: Live/Dead fixable Aqua Dead Cell Stain Kit (Invitrogen).

Regli I.B., Fernández O.L., Martínez-Salazar B., Gómez M.A., Saravia N.G, & Tacchini-Cottier F. (2018). Resistance of Leishmania (Viannia) Panamensis to Meglumine Antimoniate or Miltefosine Modulates Neutrophil Effector Functions. Frontiers in Immunology, 9, 3040.

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