For staining surface markers, cells were washed and blocked in FACS buffer (PBS plus 1% FCS) in the presence of Fc block. Samples were stained with anti-CD8α (clone 53–6.7, BioLegend), anti-CD25 (clone 7D4, eBioscience), anti-CD62L (clone MEL-14, eBioscience), anti-CD69 (clone H1.2F3, eBioscience), anti-Vα2 (clone B20.1, BD Biosciences), anti-CD244 (clone C9.1, BD Biosciences), anti-Ly108 (clone 13G3, BD Biosciences), or anti-CD27 (clone LG.3A10, BioLegend), at 4 C for 30 minutes in FACS buffer, protected from light, followed by fixation with 4% paraformaldehyde (PFA, Electron Microscopy Sciences). For intracellular staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) at 4 C for one hour. Samples were then stained with anti-granzyme B (clone GB11, BD Biosciences) for one hour at 4 C, protected from light. For phospho-antibody staining, cells were fixed with 4% paraformaldehyde, methanol-permeabilized at −20 C, and stained for 60 minutes at 4 C with anti-phosphoS6 (clone D57.2.2E, Cell Signaling) in PBS plus 1% Triton X-100 and 0.5% bovine serum albumin (BSA). Data were acquired on either a Calibur1 or LSRII flow cytometer (BD) and analyzed using FlowJo software (Tree Star).
Anti cd62l clone mel 14
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD62L (clone MEL-14) is a laboratory reagent used for the detection and analysis of CD62L (L-selectin) expression on cell surfaces. It is a monoclonal antibody that specifically binds to the CD62L antigen, which is involved in the homing and migration of lymphocytes. The core function of this product is to facilitate the identification and quantification of CD62L-positive cells in various research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd69 clone h1.2f3, by Thermo Fisher Scientific (2 mentions)
Anti cd8α clone 53 6, by BioLegend (1 mentions)
Anti cd27 clone lg 3a10, by BioLegend (1 mentions)
Anti granzyme b clone gb11, by BD (1 mentions)
Anti phosphos6 clone d57.2.2e, by Cell Signaling Technology (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti cd44 clone im7, by BioLegend (1 mentions)
Anti cd4 clone gk1.5, by BD (1 mentions)
Anti cd8 clone 53 6, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti f4 80 clone bm8, by BioLegend (1 mentions)
Anti cd8α clone 53 6, by BD (1 mentions)
Anti iaie clone m5 114, by BioLegend (1 mentions)
Anti mouse cd16 32 clone 2.4g2, by BD (1 mentions)
Anti ki67 clone sola15, by Thermo Fisher Scientific (1 mentions)
Anti cd3 clone 17a2, by BioLegend (1 mentions)
Anti cd4 clone rm4 5, by BD (1 mentions)
Anti cd44 clone im7, by BD (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
4 protocols using anti cd62l clone mel 14
1
Flow Cytometric Analysis of Immune Cell Markers
2
Lymph Node Immune Cell Profiling
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Popliteal and inguinal lymph nodes were harvested and single-cell suspensions were prepared by mechanical dissociation on a cell strainer (RPMI-1640 with 10% FBS). Cell suspensions were centrifuged at 300g for 5 min. Erythrocytes in lymph nodes were lysed with ammonium-chloride-potassium lysis buffer for 5 min at RT. Cell suspensions were washed and filtered through 40-μm filters. Nonspecific staining was reduced by using Fc receptor block (anti-CD16/CD32). Cells were incubated for 30 min with varying combinations of the following fluorophore-conjugated monoclonal antibodies: anti-CD3e (clone 145-2C11; BD pharmigen), anti-CD4 (clone GK1.5; BD Pharmingen), anti-CD8 (clone 53-6.7; eBioscience), anti-CD44 (clone IM7; BioLegend), and anti-CD62L (clone MEL-14; eBioscience) antibodies (diluted at a ratio of 1:200) in FACS buffer (5% bovine serum in PBS). After several washes, cells were analysed by FACS Canto II (BD Biosciences) and the acquired data were further evaluated by using FlowJo software (Treestar).
3
Multiparameter Flow Cytometry of Immune Cells
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Single cell suspensions (1 × 106 cells/100 μl) were prepared with dead cells excluded by using Zombie Aqua Fixable Viability Kit (Biolegend, USA). After being washed, cells were resuspended in buffer (PBS containing 5% FBS) and were blocked in Fc block (Anti-mouse CD16/32, Clone 2.4G2, BD, USA) at 4°C for 30 min. Cells were then incubated with following various antibodies for 30 min at 4°C. anti-CD11b (clone M1/70, Biolegend, USA), anti-F4/80 (clone BM8, Biolegend, USA), anti-Ly-6G/Ly-6C (clone RB6-8C5, eBioscience, USA), anti-CD11c (clone N418, Biolegend, USA), anti-I-A/I-E (clone M5/114.15.2, Biolegend, USA); Rat IgG2b,ҝ Isotype Ctrl (Biolegend, USA); anti-CD3 (clone 17A2, Biolegend, USA), Anti-CD4 (clone RM4-5, BD, USA), anti-CD8α (clone 53–6.7, BD, USA), anti-CD44 (clone IM7, BD, USA); anti-CD62L (clone MEL-14, eBioscience, USA), anti-Ki67 (clone SolA15, eBioscience, USA). Ki67 staining was implemented using Foxp3/Transcription Factor Staining Buffer Set (eBioscience, USA) according to manufacturers’ instructions.
4
Flow cytometry analysis of T cell activation
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Cell viability was determined by Fixable Viability Dye eFluor 506 (eBioscience). Cells were subsequently washed (400 x g for 5 minutes) in FACS buffer and incubated with Fc Block for 15 minutes at 4 °C prior to addition of fluorochrome-conjugated antibodies (30 minutes at 4 °C).
Antibodies used (eBioscience): anti-CD3 (clone 17A2); anti-CD4 (clone GK1.5); anti-CD45 (clone 30-F11); anti-CD45.1 (clone A20); anti-CD69 (clone H1.2F3); anti-CD44 (clone IM7); anti-ICOS (clone 7E.17G9); anti-CD62L (clone MEL-14); anti-CD19 (clone eBio1D3); anti-MHC-II (I-A/I-E) (clone M5/114.15.2); anti-CD11c (clone N418). For fluorescence-activated cell sorting (FACS), joint draining popliteal LNs were pooled on day 4 post-HAO challenge and stained for viability, anti-CD45, anti-CD3 and anti-CD4. Live CD45 + CD3 + CD4 + T cells were isolated using a FACS Aria IIU (BD Biosciences, Oxford, UK) and co-cultured with BMDC at a 1:10 ratio for 72 hours at 37 °C and 5% CO 2 . Intracellular cytokine staining was performed on cells isolated from joints and draining LNs using anti-IFNg (XMG1.2) and anti-TNFa (MP6-XT22) following stimulation with either OVA 323-339 (0.5 µg/mL) or PMA (10 ng/ml) and ionomycin (500 ng/ml) for 4 hours in the presence of brefeldin A. Cells were acquired on an LSRII (BD Biosciences) and data analysed using FlowJo software (TreeStar version 7.6.5, Oregon, USA).
Antibodies used (eBioscience): anti-CD3 (clone 17A2); anti-CD4 (clone GK1.5); anti-CD45 (clone 30-F11); anti-CD45.1 (clone A20); anti-CD69 (clone H1.2F3); anti-CD44 (clone IM7); anti-ICOS (clone 7E.17G9); anti-CD62L (clone MEL-14); anti-CD19 (clone eBio1D3); anti-MHC-II (I-A/I-E) (clone M5/114.15.2); anti-CD11c (clone N418). For fluorescence-activated cell sorting (FACS), joint draining popliteal LNs were pooled on day 4 post-HAO challenge and stained for viability, anti-CD45, anti-CD3 and anti-CD4. Live CD45 + CD3 + CD4 + T cells were isolated using a FACS Aria IIU (BD Biosciences, Oxford, UK) and co-cultured with BMDC at a 1:10 ratio for 72 hours at 37 °C and 5% CO 2 . Intracellular cytokine staining was performed on cells isolated from joints and draining LNs using anti-IFNg (XMG1.2) and anti-TNFa (MP6-XT22) following stimulation with either OVA 323-339 (0.5 µg/mL) or PMA (10 ng/ml) and ionomycin (500 ng/ml) for 4 hours in the presence of brefeldin A. Cells were acquired on an LSRII (BD Biosciences) and data analysed using FlowJo software (TreeStar version 7.6.5, Oregon, USA).
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