Antibodies used include: Ii(In-1, 1:100), H2-M (2E5A, 1:50), Rat IgG1 (R3–34, 1:50) (all from BD Biosciences); podoplanin (8.1.1, Biolegend, 1:500); CLIP (15G4, Santa Cruz, 1:5); H2-O69 (link) (Mags.Ob3, Lisa Denzin, 1:400); 10.1.1 (UVA lymphocyte culture center, 1:1000); CD45 (30-F11, 1:1000), CD31 (390, 1:1000), MHC-II (M5/114.15.2, 1:50 (Fig 1 ) to 1:500 (Fig 6 )), CD8 (53-6.7, 1:1000), CD4 (GK1.5, 1:1000), Thy1.1 (HIS51, 1:1000), Thy1.2 (53-2.1, 1:1000), CD45.1 (A20, 1:500), CD11c (N418, 1:500), CD11b (M1/70, 1:500), CD69 (H1.2F3, 1:500), CD62L (MEL-14, 1:1000), CD44 (IM7, 1:1000), CD25 (PC61.5, 1:500), Y-AE (eBioY-Ae, 1:200), PD-1 (RMP1–30, 1:100), LAG-3 (eBioC9B7W, 1:100), Rat IgG2b (eB149/10H5) (all from eBioscience). Intracellular staining for PD-1, LAG-3, Ii, H2-M and H2-O was done using the Cytofix/Cytoperm kit (BD Bioscience), and Ki67 (SolA15, 1:500) and FoxP3 (FJK-16s, 1:100) were stained using Treg permeabilization buffers (eBioscience). Annexin V (1:20) was stained using the eBioscience kit. DAPI (Sigma) or live/dead aqua (Invitrogen, 1:200) were used to distinguish live cells. Cells were acquired on a FACSCanto II (BD Biosciences) and data analyzed using FlowJo (Tree Star).
Cd62l
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
CD62L is a cell surface glycoprotein that functions as a cell adhesion molecule. It is expressed on the surface of various cell types, including leukocytes, and plays a role in the initial tethering and rolling of these cells on the endothelium during the inflammatory response.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by Thermo Fisher Scientific (9 mentions)
Ifn γ, by Thermo Fisher Scientific (9 mentions)
Facscanto 2, by BD (5 mentions)
F4 80, by Thermo Fisher Scientific (4 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (4 mentions)
Lsrii flow cytometer, by BD (4 mentions)
Ionomycin, by Merck Group (4 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Il 17a, by Thermo Fisher Scientific (3 mentions)
Facsdiva software, by BD (3 mentions)
H 2kb, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (3 mentions)
Facsaria, by BD (2 mentions)
F4 80, by Bio-Rad (2 mentions)
Fluorescence activated cell sorting (facs), by Beckman Coulter (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Gata3, by Thermo Fisher Scientific (2 mentions)
Ter119, by Thermo Fisher Scientific (2 mentions)
Fluorophore conjugated icos, by Thermo Fisher Scientific (2 mentions)
R phycoerythrin affinipure f ab 2 fragment donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
58 protocols using cd62l
1
Comprehensive Immune Cell Phenotyping
2
Murine Immune Cell Profiling
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Single-cell suspensions of bone marrow, spleen, or peritoneal fluid were prepared and red blood cells were lysed. All analyzed mice were 6–12 weeks of age, unless indicated differently. Cells were incubated with various combinations of the following antibodies: CD3, CD4, CD5, CD8, CD19, CD21, CD23, CD24, CD62L, B220, AA4.1, CXCR5, FAS, GL7, Gr-1, IgD, IgM, PD1, and TCR-β (eBioscience and Tonbo). Samples were collected using FACS LSRII (BD) or sorted using FACSAria (BD), and data were analyzed with FlowJo software (Tree Star). Differences between groups were analyzed using unpaired t-test analysis (GraphPad Prism software).
3
Multiparametric Flow Cytometry Analysis
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For flow cytometric analysis, blood was obtained by cardiac puncture with an EDTA-coated syringe. Spleen and LNs were removed and collected in phosphate-buffered saline (PBS). EpAT and ScAT were prepared separately by mincing into small pieces and digested using liberase (0.25 mg/mL Dulbecco’s Modified Eagle Medium, Roche) for 45 min at 37°C. Spleen, LN, and digested adipose tissue samples were passed through a 70 µm nylon mesh (BD Biosciences). The stromal vascular fraction (SVF) was collected by pelleting of the suspension and diluted in fluorescence-activated cell sorting (FACS) buffer (0.5% bovine serum albumin, 0.01% NaN3 and 2 mM EDTA in PBS). Blood and spleen were incubated with hypotonic lysis buffer (8.4 g NH4Cl and 0.84 g NaHCO3 in 1 L miliQ) to remove erythrocytes. Cell suspensions were blocked with CD16/32 antibody (eBioscience, Waltham, Massachusetts, USA) in FACS buffer to prevent non-specific binding. Indicated tissues were incubated with CD45, CD19, CD62L, CD44, FoxP3, Ly6C (eBioscience), CD3, CD8, CD38, CXCR3, CD40L (Biolegend, San Diego, California, USA), CD4, CD11b, Ly6G, SiglecF (BD Biosciences), and F4/80 (AbD serotec) antibodies. Fluorescence was measured by flow cytometry (FACSCanto II, BD Biosciences) and analysed with FlowJo software V.10 (Tree star).
4
Single-Cell Analysis of Cardiac Immune Cells
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Single‐cell suspensions were pooled from heart tissues. Surface markers were stained with fluorochrome‐conjugated mAbs diluted in 1% FBS in PBS: CD4, CD62L, F4/80, Arg1 and iNOS (eBioscience; BD Pharmingen; Biolegend). For intracellular staining, cells were fixed and permeabilized using fixation buffer and permeabilization solution (eBioscience). Cell fluorescence was measured using FACS (Beckman Coulter), and data were analysed using FlowJo software (Treestar).
5
Cell Surface Marker Analysis in Thymus and Spleen
6
Multiparameter Flow Cytometry
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Fluorophore-conjugated ICOS, PD1, CXCR5, CD4, CD8, TCRb, CD44, CD62L, CD25, CD69, CD5, CD11b, CD11c, CD19, B220, Ter119, DX5, Gr1, IFNγ, IL-4, IL-17A, FoxP3, Gata3, and Bcl6 were purchased from eBioscience, BD Biosciences, BioLegend, and Tonbo Biosciences. Primary antibodies for phospho-flow cytometry were purchased from Cell Signaling Technologies: P-S6 S235/236 (2211), P-S6 S240/244 (2215), P-ERK (4377), and P-Akt (4058). Secondary antibody for phospho-flow cytometry was R-Phycoerythrin AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG (Jackson Immunoresearch).
7
Flow Cytometric Analysis of Immune Cells
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The following fluorescent mouse Abs from Biolegend (San Diego, CA, USA) were used for flow cytometry analysis: CD3, CD4, CD8, CD103, CD25, CD62L, CD69, CD86, CD138, Foxp3, and Ki-67; from eBioscience (San Diego, CA, USA): B220, granzyme B, perforin; from Santa Cruz (Dallas, TX, USA): granzyme A. Cell subsets were stained with mAbs and isotype control as indicated above and analyzed on BD LSRFortossa™ flow cytometer (BD Biosciences, San Diego, CA, USA) using FACSDiva Software (BD Biosciences). For intracellular staining, such as Foxp3, Granzyme A, GranzymeB, perforin, and Ki-67, cells were first stained with surface marker, and further fixed and permeabilized for intracellular staining using Fix and Perm (eBioscience). Final plmiceot/histogram figures were prepared using FlowJo Software (Tree Star, Ashland, OR, USA).
8
Multiparameter Flow Cytometry
9
Synovial Fluid Leukocyte Profiling
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After lysis of red blood cells, total synovial fluid leukocytes were counted in a Neubauer chamber under microscopy (×10 objective). Single-cell suspensions were prepared, washed and resuspended in flow cytometry buffer ((2% foetal calf serum (Invitrogen, Sao Paulo, Brazil) in PBS (Sigma-Aldrich, Sao Paulo, Brazil), and stained with different monoclonal antibodies conjugated to fluorochromes, for 30 min: Ly6C and CD19 (FITC); Ly6G and CD3 (PE); CD11b and CD4 (PerCP); CD62L, CD14 and CD3 (APC), all from eBiosciences (Sao Paulo, Brazil). Data was collected in a BD Accuri C6 Flow Cytometer (BD Biosciences-Immunocytometry Systems, San Jose, CA, USA). The events were analysed with FlowJo 7.6.1 software (TreeStar-Ashland, OR, USA). Results are expressed as absolute cell counts or relative numbers; excepting those of CD62L and CD11b which were expressed as mean fluorescence.
10
Multiparameter Immune Cell Profiling
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After a 2.4G2 blocking step, cells were stained for CD4, CD8, CD44, CD62L, CD69, B220, CD25, CD3, CD127, and Vβ2 or Vβ14 (eBioscience or Biolegend or BD-Pharmingen, San Diego, CA, USA). All antibody incubations were performed at 4°C for 30 minutes (isotype controls were included). Intracellular Foxp3 staining was performed using Foxp3 staining kit (eBioscience) and manufacturer's protocol. Intracellular cytokine staining was performed as described [13] . Cells were immediately acquired on a LSRII flow cytometer (BD Biosciences) and analyzed using the FlowJo software (Treestar).
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