Abs directed against phosphotyrosine (PY1000), phospho–spleen tyrosine kinase (Syk) (Y525/526), phospho-pan Src (Y527 and Y416), phospho-Akt (S473), phospho-p38 (T180, Y182), and phospho-Erk (T202, Y204) were from Cell Signaling Technology. Anti-Syk (clone 5F5) and anti-Lyn (clone LYN-01) were from BioLegend. Anti-Ly6G (clone RC6-8C5) was obtained from R&D Systems. Anti-BSA and anti-lactoferrin Abs were from Sigma and an Ab against β-COP was a gift from Nick Ktistakis (The Babraham Institute, Cambridge, U.K.). HRP-conjugated secondary Abs were from Santa Cruz Biotechnology and Bio-Rad. Fluorescently conjugated Abs for flow cytometry were obtained from eBioscience (F4/80, GR1), BioLegend (CD11b, CD11a, CD16/32, Ly6G, CD62L, CD19), and BD (Ly6C).
Cd11a
Manufactured by BD
Sourced in United States
CD11a is a cell surface receptor that belongs to the integrin family of proteins. It is expressed on the surface of various immune cells, such as leukocytes, and plays a role in cell adhesion and migration. The primary function of CD11a is to facilitate the attachment and interaction of cells with other cells or the extracellular matrix.
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Lab products found in correlation
Hla dr, by BD (3 mentions)
Cd11b, by BD (2 mentions)
Cd106, by BD (2 mentions)
Phospho erk t202 y204, by Cell Signaling Technology (1 mentions)
Anti syk clone 5f5, by BioLegend (1 mentions)
Anti lyn clone lyn 01, by BioLegend (1 mentions)
Anti ly6g clone rc6 8c5, by R&D Systems (1 mentions)
Transwell system, by Corning (1 mentions)
Cd162, by BD (1 mentions)
Formyl methionyl leucyl phenylalanine (fmlp), by Merck Group (1 mentions)
Anti mouse igg fitc, by Merck Group (1 mentions)
Delta1, by Santa Cruz Biotechnology (1 mentions)
Pdgf bb, by Merck Group (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg fluorescein isothiocyanate fitc, by Merck Group (1 mentions)
Hla abc, by BD (1 mentions)
Cd166, by BD (1 mentions)
Cd44v6, by BD (1 mentions)
Hla class 1, by BD (1 mentions)
Anti cd10, by BD (1 mentions)
8 protocols using cd11a
1
Multiparameter Phosphoprotein Analysis
2
Transwell Migration Assay for PMN Infiltration
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Human epithelial cells were cultivated on inserts of a Transwell system (Costar, MA, USA) until reaching confluence. Thereby, cells were seeded on the bottom side of the inserts, so that cells migrate—comparable to the in vivo migration—from the basolateral side through the epithelial monolayer. PMNs from whole human blood migrated through a monolayer of epithelial cells along a chemotactic gradient (fMLP; 20 ng/ml; Sigma-Aldrich, Steinheim, Germany) (Supplemental Figure 3 ). After 4 h, PMNs in the upper chamber and also migrated PMNs in the lower chamber were collected. To determine PMNs in the epithelial layer, wells were treated with 800 μl Accutase and incubated and washed with 2 ml medium. Absolute cell counts were determined. PMNs were identified CD45 (B183057, BioLegend) and CD66b (E16847-109 eBioscience) positive, adhesion molecules by: CD11a (catalog number 3189709, BD), CD11b (54342, BD), CD31 (4065855, BD), CD54 (4301917, BD), CD162 (61514, BD), CD47 (E15361-103, eBioscience), CD172a (3290897, BD), CD44 (B180554, BioLegend), and CD29 (4262918, BD). As corresponding IgG controls were IgG1 (16651, BD) and IgM (E13114-104, eBioscience) used.
3
Characterization of Mesenchymal Stem Cells
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AMSCs were grown in Dulbecco’s modified Eagle’s medium (L-DMEM), IMDM, DF12 (Life Technologies, San Diego, CA, USA), with platelet-derived growth factor BB (PDGF-BB, Sigma, St. Louis, MO, USA). The following antibodies were used: CD11a, CD29, CD31, CD34, CD44, CD45, CD73, CD105, CD106, CD166, CD184, HLA-ABC, HLA-DR (BD Biosciences, San Jose, CA, USA); anti-rabbit IgG- fluorescein isothiocyanate (FITC; Sigma), anti-mouse IgG-FITC (Sigma). Jagged-1 (Calbiochem, San Diego, CA); Jagged-2 (Chemicon, Temecula, CA); Delta-1(Santa Cruz Biotechnology, CA).
4
Blocking ICAM-1 and LFA-1 in CD4+ T cell activation
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Blocking antibodies against ICAM-1 (CD54) and LFA-1 (CD11a), and matched isotype control IgG1 (all BD Biosciences) were added to the CD4+ T cell/vaccine cell co-cultures at boosting in a concentration range of 0,2-5μg/ml. Mel202 and Mel202/DR1/CD80 cells were analyzed for ICAM-1 expression, and vaccine-activated CD4+ T cells were analyzed for LFA-1 expression by flow cytometry.
5
Characterization of Cell Surface Markers
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The following fluorescein (FITC)-, allophycocyanin (APC)-or phycoerythrin (PE)-conjugated mouse anti-human monoclonal antibodies (mAbs) were used: Anti-CD10, -CD11a, CD11c, -CD18, -CD33, -CD40, -CDD44std, -CD44v5, -CD44v6, -CD54, -CD61, -CD62P, -CD86, -CD133, -CD206 (MR), -EGFR, -Her-2/neu, -HLA-DR, HLA class I, -CCR5, -CCR6, Her-2/neu all from BD Pharmingen (San Diego, CA, USA); anti-CD29, -CD36, -CD51, -CD58 from Immunotech (Marseille, France); anti-c-MET, -CCR1, -CCR2, -CCR3, -CCR7, -CXCR1, -CXCR2, -CXCR4 from R&D (Abington, UK); anti-Tag72, -Mucin1 (EMA, CD227), -EMMPRIN from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and anti-Epithelial Antigen, -Epithelial Membrane Antigen (EMA) from DAKO (Heverlee, Belgium). Isotype controls included appropriate FITC-, APC- or PE-labeled mouse IgG1, IgG2a or IgG2b. Cells were incubated with mAbs or isotype controls for 20 min at 4°C, washed, resuspended in PBS and analyzed by flow cytometry (FACS Canto; BD Biosciences Immunocytometry Systems, San Jose, CA, USA) using FACS DiVa software.
6
Investigating Immune Cell Signaling Pathways
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The following antibodies were used as follow: For flow cytometry determination, CD3, CD8, CD54 (intercellular adhesion molecule [ICAM]-1), CD106 (vascular adhesion molecule [VCAM]-1), CD11a (lymphocyte function-associated antigen-1 [LFA-1]), CD29 (integrin β1), IL-27, IFN-γ, IL-17, phosphorylated (p)-STAT1 (pY701), p-STAT1 (pS727), p-STAT2 (pY689), p-STAT3 (pS727), p-STAT3 (pY705), p-STAT4 (pY693), p-STAT5 (pY694), p-STAT6 (pY641), TCCR-WSX-1 (IL-27 receptor), IL-17 receptor (R), IFN-γR1, and Ki-67 mAbs were purchased from BD Biosciences (Franklin Lakes, USA), eBioscience (San Diego, USA), or R and D systems (Minneapolis, USA); for cytokine neutralization, IL-27, IFN-γ, IL-17, ICAM-1 and VCAM-1 mAbs were from eBioscience.
Recombinant human IL-27, IFN-γ, IL-17A were purchased from R and D systems or eBioscience. STAT1 inhibitor S14-95 and STAT3 inhibitor Galiellalactone were from Enzo Life Sciences (Farmingdale, USA). Annexin V Apoptosis Detection Kit was from eBioscience. PMA and ionomycin were from Sigma-Aldrich (St. Louis, USA), and GolgiPlug and GolgiStop were from BD Bioscience. Foxp3 fixation/permeabilization concentrate and diluent were from eBioscience. Fluorescent dye CFSE was from Invitrogen (Carlsbad, USA).
Recombinant human IL-27, IFN-γ, IL-17A were purchased from R and D systems or eBioscience. STAT1 inhibitor S14-95 and STAT3 inhibitor Galiellalactone were from Enzo Life Sciences (Farmingdale, USA). Annexin V Apoptosis Detection Kit was from eBioscience. PMA and ionomycin were from Sigma-Aldrich (St. Louis, USA), and GolgiPlug and GolgiStop were from BD Bioscience. Foxp3 fixation/permeabilization concentrate and diluent were from eBioscience. Fluorescent dye CFSE was from Invitrogen (Carlsbad, USA).
7
Phenotypic Characterization of hUC-MSCs
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Flow cytometry was used to analyze the phenotype of hUC-MSCs. Trypsinized cells were suspended in phosphate-buffered saline (catalog no. PBS-10001, Cyagen Biosciences, Inc.) at a density of 5 × 106 cells/ml, and a 100 μl sample was incubated with various BD Pharmingen™ PE mouse anti-human CD73 (catalog no. 561014, 1 : 50), CD105 (catalog no. 560839, 1 : 50), CD34 (catalog no. 560941, 1 : 50), and BD Pharmingen™ FITC mouse anti-human CD90 (catalog no. 561969, 1 : 50), CD45 (catalog no. 560976, 1 : 50), CD11a (catalog no. 555383, 1 : 50), and HLA-DR (catalog no. 560944, 1 : 50) antibodies (BD Biosciences) for 45 min at room temperature. Control samples were incubated with PBS instead of antibodies. Antibody binding was examined using a FACScan flow cytometer (Beckman Coulter) and was analyzed using FlowJo v10.6.2 (BD Biosciences).
8
Coverslip Preparation for Imaging Spread Cells
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The preparation of coverslips for imaging spread cells followed a previously described technique (e.g. 2 ). Briefly, for diffraction limited and PALM imaging, coverslips (#1.5 glass chambers, LabTek or iBidi) were washed with acidic ethanol at room temperature (RT) for 10 min; liquid was then aspirated and coverslips were dried at 40°C for 1 hour.
Cleaned coverslips were incubated at RT for 15 min with 0.01% poly-L-lysine (Sigma) diluted in water. Liquid was aspirated and coverslips were dried at 40°C for 12 hours.
Coverslips were subsequently incubated with stimulatory or non-stimulatory antibodies at a concentration of 10 mg/ml (unless specified otherwise) overnight at 4°C or 2 hours at 37°C. Finally, coverslips were washed with PBS. Throughout the study we used the following stimulatory antibodies: purified mouse anti human CD3 (clone Ucht1) and CD11a (BD Biosciences). A few minutes before imaging, cells were resuspended in imaging buffer at a concentration of 1 million/150 ml and 100,000-500,000 cells were dropped onto coverslips for PALM or diffraction limited imaging, incubated at 37°C for the specific spreading time (typically 3 min) and fixed with 2.4% PFA for 30 min at 37°C or used for live cell imaging.
Cleaned coverslips were incubated at RT for 15 min with 0.01% poly-L-lysine (Sigma) diluted in water. Liquid was aspirated and coverslips were dried at 40°C for 12 hours.
Coverslips were subsequently incubated with stimulatory or non-stimulatory antibodies at a concentration of 10 mg/ml (unless specified otherwise) overnight at 4°C or 2 hours at 37°C. Finally, coverslips were washed with PBS. Throughout the study we used the following stimulatory antibodies: purified mouse anti human CD3 (clone Ucht1) and CD11a (BD Biosciences). A few minutes before imaging, cells were resuspended in imaging buffer at a concentration of 1 million/150 ml and 100,000-500,000 cells were dropped onto coverslips for PALM or diffraction limited imaging, incubated at 37°C for the specific spreading time (typically 3 min) and fixed with 2.4% PFA for 30 min at 37°C or used for live cell imaging.
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