Cd62l pe cy7

Manufactured by BioLegend

Sourced in United Kingdom, United States

CD62L-PE/Cy7 is a flow cytometry reagent that binds to the CD62L (L-selectin) antigen. CD62L is a cell surface adhesion molecule expressed on various leukocyte subsets. This reagent can be used to identify and characterize cells expressing CD62L.

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Lab products found in correlation

Cd4 fitc, by BioLegend (4 mentions) Cd44 apc cy7, by BioLegend (3 mentions) Cd3 alexa fluor 700, by BioLegend (3 mentions) Cytoflex, by Beckman Coulter (3 mentions) Foxp3 pe, by Thermo Fisher Scientific (2 mentions) Granzyme b pe, by BioLegend (2 mentions) Il 4 apc, by BioLegend (2 mentions) Cd45 apc, by BioLegend (2 mentions) Cd8 apc, by BioLegend (2 mentions) Ifn γ pe cy7, by BioLegend (2 mentions) Ionomycin, by Merck Group (2 mentions) Cd62l pe, by BD (2 mentions) Cd4 pe fitc apc, by BD (2 mentions) 41bb apc pe, by BD (2 mentions) Cd8 pe cy7 fitc, by BD (2 mentions) Ox40 fitc pe cy7, by BD (2 mentions) Cd45ro apc bv421, by BD (2 mentions) Sh800s ma900 instrument, by Sony (2 mentions) Cd8a efluor 450, by Thermo Fisher Scientific (2 mentions) Facscanto 3, by BD (2 mentions)

Close spelling variants of this product

PE-Cy7-anti-CD62L PE-Cy7-labeled anti-CD62L PE/Cy7 anti-mouse CD62L PE-Cy7-conjugated anti-mouse CD62L ( CD62L-PE CD62L

22 protocols using cd62l pe cy7

T-cell Immunophenotyping by Flow Cytometry

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CAR moiety was labeled with a donkey anti-mouse Fab (Abcam, 150109). For T-cell phenotyping, the following antibodies were used: mouse anti-human PE-CD8 (Clone HIT8a, 12-0089-42), APC-CD56 (Clone TULY56, 17-0566-42) from eBiosciences; mouse anti-human APC-CD279 (Clone EH12.2H7, 329908), FITC-CD223 (Clone 11C3C65, 369308), Brilliant Violet 421-CD127 (Clone A019D5, 351310), PE/cy7-CD62L (Clone DREG-56, 304822), PE-biotin (Clone 1D4-C5, 409003) from Biolegend; and mouse anti-human Brilliant Violet 421-Tim-3 (Clone 7D3 (RUO), 565562) from BD Horizon. For intracellular staining, T cells were fixed and permeabilized using BD Cytofix/Cytoperm kit as the recommendation of the manufacturer. Anti-human CD8-PE (Clone HIT8a, eBiosciences, 12-0089-42) and donkey anti-mouse Fab (Polyclonal, Abcam, ab150109) were used for extracellular staining, and anti-human IFN-γ-eFluor 450 (Clone 4 S.B3, 85-48-7319-42, eBiosciences) was used for intracellular staining. Stained samples were acquired on a BD FACSAria and analyzed with FlowJo software (Tree Star, Ashland, OR).

Zou F., Lu L., Liu J., Xia B., Zhang W., Hu Q., Liu W., Zhang Y., Lin Y., Jing S., Huang M., Huang B., Liu B, & Zhang H. (2019). Engineered triple inhibitory receptor resistance improves anti-tumor CAR-T cell performance via CD56. Nature Communications, 10, 4109.

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Multi-Color Flow Cytometry Analysis of Murine Immune Cells

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All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target” format as follows: FITC-IL-17A, PE-Foxp3, Allophycocyanin-IFN-γ, Allophycocyanin-Cy7-CD4, PerCP-Cy5.5-CD25, PE-Cy5-ICOS, PerCP-eFluor 710-TNF-α, and PE-Cy7-Thy1a were from eBioscience (San Diego, CA); FITC-CD25, FITC-CD45.1, FITC-Thy1a, PECF594-CD4, PE-CF594-CD8α, Allophycocyanin-ICOS, Alexa Fluor 700-CD45.2, PECy7-CD4 and Allophycocyanin-Cy7-TCRβ were from BD Biosciences (San Diego, CA); Alexa Fluor 610-CD4 and PE-Texas Red-CD8α were from Invitrogen (Carlsbad, CA); Brilliant Violet 421-NRP1, Alexa Fluor 700-CD45.1 and PE-Cy7-CD62L were from Biolegend (San Diego, CA), Foxp3 staining buffer kit was from eBiosciences. To detect cytokines, cells were stimulated with PMA/Ionomycin and analyzed as previously described (33 ).

Huang W., Jeong A.R., Kannan A.K., Huang L, & August A. (2014). IL-2 inducible T cell kinase (ITK) tunes T regulatory cell development and is required for suppressive function. Journal of immunology (Baltimore, Md. : 1950), 193(5), 2267-2272.

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Peptide Library Synthesis and Liposome Formulation

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Trp2, AH1 and WT1 peptide libraries and peptides were synthesized by GenScript. CoPoP was synthesized as previously reported (32 (link)). The following lipids were used to produce liposomes: dioleoylphosphatidylcholine (DOPC; Corden #LP-R4–070), cholesterol (PhytoChol; Wilshire), and synthetic PHAD-3D6A (Avanti; #699855). QS-21 was purchased from Desert King (part number NC0949192). The APC-CD8a (Clone: CT-CD8a) antibody was obtained from Accurate Chemical and Scientific Corporation (#ACL168APC). The following antibodies were obtained from BioLegend: APC-CD8a (Clone: CT-CD8a; #100712), FITC-CD4 (Clone: GK1.5; #100405), AF700-CD45 (Clone: 30-F11; #103127), APC-Cy7-CD44 (Clone: IM7; #103027), PE/Cy7-CD62L (Clone: MEL-14; #104417), PerCP-Cy5.5-PD-1 (Clone: RMP1–30; #109119), pacific blue-IFN-γ (Clone: XMG1.2; #505818), BV605-TNF-α (Clone: MP6-XT22; #506329), PE/Cy7-Granzyme B (Clone: QA16A02; #372213). Anti-mouse PD-1 (#BP0146) and anti-mouse CTLA-4 (#BP0131) were acquired from Bio X Cell. Other reagents used were Golgiplug/Brefeldin A (BD; #555029), Live/Dead dye (Invitrogen; #L34857), Fc-block (Clone: 2.4G2; BD #553142), fixation/permeabilization kit (BD #554714), red blood cell (RBC) lysis buffer (BioVision #5830), Collagenase Type I (Gibco #17018–029), and DNase I (Roche #04536282001).

He X., Zhou S., Quinn B., Jahagirdar D., Ortega J., Long M.D., Abrams S.I, & Lovell J.F. (2022). An In Vivo Screen to Identify Short Peptide Mimotopes with Enhanced Antitumor Immunogenicity. Cancer immunology research, 10(3), 314-326.

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Multi-parameter Immune Cell Profiling

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Antibodies for APC-CD3, PE-CD4, PE/Cy7-CD8, Alexa Fluor647-CD56, PE-CD107a, PE-CCR7, PE/Cy7-CD62L, PE/Cy7-perforin, PE-granzyme B, PE-PD-L1 were purchased from BioLegend. Goat anti-B7-H3 antibody (MAB1027) was purchased from R&D System. Humanized anti-B7-H3 IgG (8H9) was expressed and purified in the laboratory (for details, see Additional file 1). Mouse anti-CD16 antibody (3G8) was purchased from BD Biosciences. PE-conjugated anti-human Fc antibody and HRP anti-human IgG antibody were purchased from Invitrogen. HRP-conjugated rabbit anti-goat IgG antibodies were purchased from Jackson ImmunoResearch. Rabbit antihuman PD-L1 antibody (13,684), HRP-conjugated anti-β-actin and anti-GAPDH antibodies were purchased from Cell Signaling.

Liu J., Yang S., Cao B., Zhou G., Zhang F., Wang Y., Wang R., Zhu L., Meng Y., Hu C., Liang H., Lin X., Zhu K., Chen G., Luo K.Q., Di L, & Zhao Q. (2021). Targeting B7-H3 via chimeric antigen receptor T cells and bispecific killer cell engagers augments antitumor response of cytotoxic lymphocytes. Journal of Hematology & Oncology, 14, 21.

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Multiparametric Flow Cytometry Analysis

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Single-cell suspensions were prepared from the spleen and VAT and stained with PE-MHC-II (107613, Biolegend), APC-cy7-CD11c (117323, Biolegend), FITC-CD64 (139315, Biolegend), Percp-cy5.5-CD45.2 (109827, Biolegend), APC-CD45 (147707, Biolegend), FITC-CD4 (100405, Biolegend), APC-CD8 (126613, Biolegend), PE-cy7-CD62L (104417, Biolegend), Percp-cy5.5-CD44 (103031, Biolegend), APC-cy7-FVD, or Pacific blue-DAPI. Intracellular staining was performed using PE-Foxp3 (12-5773-82, eBioscience) and antibodies against cytokines, including BV421-CD8, PE-cy7-IFN-γ (505825, Biolegend), Percp-cy5.5-IL-17A (506919, Biolegend), and APC-IL-4 (504105, Biolegend). Cells from the spleen and VAT were treated with phorbol myristate acetate (PMA, 50 ng/mL) and ionomycin (500 ng/mL) (Sigma-Aldrich) for 4.5 h. Then the cytokines in specific cell populations were analyzed. The cells were stained at 4 °C for 30 min or 1 h for surface and intracellular staining, respectively, followed by detection using the FACS Canto II (BD Biosciences) system and analysis of the data using FlowJo (Tree Star, version 10.0).

Sun Y., Wang B., Hu Q., Zhang H., Lai X., Wang T., Zhao C., Wang J., Zhang X., Niu Q., He B., Jiang E., Shi M., Feng X, & Luo Y. (2023). Loss of Lkb1 in CD11c+ myeloid cells protects mice from diet-induced obesity while enhancing glucose intolerance and IL-17/IFN-γ imbalance. Cellular and Molecular Life Sciences, 80(3), 63.

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Multicolor Flow Cytometry Analysis of PBMC, LMNC and PMNC

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For multicolor flow cytofluorimetric analysis, PBMCs, LMNCs and PMNCs were stained with the following conjugated mAb as previously described: CD56-PE-Cy5 and NKp46-PE (Beckman Coulter, clone BAB281), CD16-PE-Cy7, CD19-APCH7, IFN-γ-PE, DNAM-1 PE, CD49e-PE, CD11c-PE, CCR5-PE, CD69-PE, CCR4-PE and CXCR3-PE (BD-Pharmigen), CD45-PB, CD62L-PE-Cy7, CCL5-AF647, CD161-PerCpCy5.5, CCR3-PE, CXCR2-PE and CXCR4-PerCpCy5.5 (Biolegend), CCR7-FITC and CXCR6-PE (R&D System), CX₃CR1 PE (MBL). Aqua LIVE/DEAD (Life Technologies) was used to eliminate dead cells from the analysis. Intracellular staining was performed using Cytofix/Cytoperm (Beckton Dickinson), according to the manufacturers instructions. The gating strategy used to select NK cells from both PBMC and LMNC is depicted in Supplemental Figure 1. For measurement of IFN-γ production, whole PBMC and LMNC were stimulated with 18 hours with 20ng/ml rhIL-12 (R&D Systems) and 200U/ml rhIL-2 (Peprotech). For the final 4 hours, GolgiStop was added (Beckton Dickenson). Flow cytometry data was acquired using an LSR Fortessa (Beckton Dickinson) and data was analyzed using FlowJo Software (Tree Star).

Hudspeth K., Donadon M., Cimino M., Pontarini E., Tentorio P., Preti M., Hong M., Bertoletti A., Bicciato S., Invernizzi P., Lugli E., Torzilli G., Gershwin M.E, & Mavilio D. (2015). Human liver-resident CD56bright/CD16neg NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways. Journal of autoimmunity, 66, 40-50.

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T Cell Phenotyping and Sorting

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The antimouse TCRβ (mTCR)- PerCp-Cy5.5, CD8a-eFluor 450, were purchased from eBioscience. CD3-APC-Cy7, CD4- PE/FITC/APC, 41BB- APC/PE, CD8- PE-Cy7/FITC, OX40- FITC/PE-Cy7, CD62L-PE, and CD45RO-APC/BV421 were purchased from BD Biosciences. CD3-Alexa Fluor 700, CD62L-PE-Cy7, and the live/dead stains- DAPI/PI were purchased from BioLegend.
For FACS sample preparation, cells were harvested and washed with FACS buffer. Cells were resuspended in FACS buffer at a concentration of 1–50 × 10⁶ cells/mL and extracellular fluorescence-conjugated primary antibodies were added and mixed in. After a 20- to 60-minute incubation at 4°C, which was also protected from light exposure, the samples' cells were washed and resuspended with FACS buffer.
The flow cytometry assays were performed on FACSCanto I/II (BD Biosciences) and the acquired data were analyzed with FlowJo software (TreeStar). Cells were sorted to: Live/CD3⁺, separated for CD4⁺ or CD8⁺, T_EMRA/T_EM/T_CM based on sorting out CD62L⁺/CD45RO⁻ (naïve T cells). Cocultures were sorted for enrichment or into single reactive cells based on 41BB⁺/OX40⁺ (both double- and single-positive cells), Live/CD3⁺, separated for CD4⁺ or CD8⁺ by SH800S/MA900 instrument (Sony Biotechnology) or by FACSAria II (BD Biosciences).

Levin N., Paria B.C., Vale N.R., Yossef R., Lowery F.J., Parkhurst M.R., Yu Z., Florentin M., Cafri G., Gartner J.J., Shindorf M.L., Ngo L.T., Ray S., Kim S.P., Copeland A.R., Robbins P.F, & Rosenberg S.A. (2021). Identification and Validation of T-cell Receptors Targeting RAS Hotspot Mutations in Human Cancers for Use in Cell-based Immunotherapy. Clinical Cancer Research, 27(18), 5084-5095.

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Multicolor Flow Cytometry Immunophenotyping

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The following antihuman antibodies were used for cell staining: CD3-PECy7 or CD3-AF700, CD4-FITC, CD8-APC, CD45-PE, CD56-PE, iNKT TCR (Vα24-Jα18 TCR)-APC, CD45RA-FITC, CD62L-PECy7, TIM3-FITC, PD1-APC, LAG3-PECy7, HLA-A2-FITC, HLA-A3-APC, HLA-B7-PECy7, and Annexin V-FITC or Annexin V-Pacific Blue and were purchased from BioLegend. Data acquisition was performed using either a BD Accuri C6 Flow cytometer (BD Biosciences) or an Attune NXT cytometer (Thermo Fisher). Flow cytometry data were analyzed using FlowJo software (Tree Star).

Fang K.K., Lee J., Khatri I., Na Y, & Zhang L. (2023). Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells. Journal for Immunotherapy of Cancer, 11(9), e007277.

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MHC Class I Tetramer Staining Protocol

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All MHC class I tetramers were made by the NIH Tetramer Core Facility. Up to five tetramer-peptide reagents with contrasting fluorescence were used in a given staining cocktail. The amount of each tetramer was titrated (0.1–1 μL) to obtain the optimal concentration for usage. Freshly isolated and 14-day cultured cells were washed with PBS, stained with tetramer cocktail in PBS + 2% FBS, and incubated at 4 °C for 30 min. An antibody cocktail comprised of CD8-PerCP/Cy5.5, CD45RA-BV510, CD62L-PE/Cy7, CD95-PE/Dazzle 594 (Biolegend) was added and samples incubated for an additional 30 min at 4 °C. Samples were then washed with FACS buffer and analyzed by flow cytometry (CytoFLEX, Beckman Coulter).

Choy C., Chen J., Li J., Gallagher D.T., Lu J., Wu D., Zou A., Hemani H., Baptiste B.A., Wichmann E., Yang Q., Ciffelo J., Yin R., McKelvy J., Melvin D., Wallace T., Dunn C., Nguyen C., Chia C.W., Fan J., Ruffolo J., Zukley L., Shi G., Amano T., An Y., Meirelles O., Wu W.W., Chou C.K., Shen R.F., Willis R.A., Ko M.S., Liu Y.T., De S., Pierce B.G., Ferrucci L., Egan J., Mariuzza R, & Weng N.P. (2023). SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors. Nature Communications, 14, 6725.

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T-cell Phenotyping by Flow Cytometry

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T cells were stained for cell-surface markers to differentiate T-cell lineage. Previous work (24 (link)) has demonstrated that CCR7, CD62L, CD45RO, and CD95 can be used to differentiate the various T-cell phenotypes by using the following expression patterns: naïve (T_N)—CCR7⁺, CD62L⁺, CD45RO⁻, CD95⁻; stem central memory (T_SCM)—CCR7⁺, CD62L⁺, CD45RO⁻, CD95⁺; central memory (T_CM)—CCR7⁺, CD62L⁺, CD45RO⁺, CD95⁺; effector memory (T_EM)—CCR7⁻, CD62L⁻, CD45RO⁺, CD95⁺; terminal effector (T_Eff)—CCR7⁻, CD62L⁻, CD45RO⁻, CD95⁺. The antibodies used for this analysis as well as quantitation of T-cell bulk described above were CD8-FITC (BD Biosciences; #347313), CD3-PE (BD Biosciences; #555340), CD4-APC (BD Biosciences; #555349), CCR7-FITC (BD Biosciences; #561271), CD95-PE (BD Biosciences; #556641), CD45RO (BD Biosciences; #559865), and CD62L-PE/Cy7 (BioLegend; 304822). Samples were then washed twice, and flow cytometry acquisition was performed on a BD FACSVerse Flow Cytometer or BD Accuri C6 (BD Biosciences).

Das R.K., Vernau L., Grupp S.A, & Barrett D.M. (2019). Naïve T-cell Deficits at Diagnosis and after Chemotherapy Impair Cell Therapy Potential in Pediatric Cancers. Cancer discovery, 9(4), 492-499.

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