Single‐cell suspensions from spleen were obtained from tissues and stained for 20 min with indicated antibodies. For intracellular staining, T cells were stimulated with eBioscience Cell Stimulation Cocktail for 6 h at 37 °C in the presence of GolgiStop. Cells were then stained with the surface marker for 15 min on ice and permeabilized using Cytofix/Cytoperm for 30 min on ice. Permeabilized cells were resuspended in Perm/Wash buffer and stained with cytokine antibody for 20 min. FACS analysis was performed with BD LSRII Flow Cytometer (BD) or Attune Flow Cytometers (ThermoFisher Scientific), and data were analyzed by BD FACSDiva, Attune NxT Software, or Flowjo software. For specific T cell analysis, splenocytes were harvested and stained with anti‐CD4, anti‐CD11a, and anti‐CD49d antibodies. Anti‐Foxp3, anti‐CD4, anti‐CD3, anti‐CD25, anti‐CD8, anti‐IFN‐γ, anti‐CD11b, anti‐Gr1, anti‐Ly6C, anti‐Ly6G, anti‐CD11a, and anti‐CD49d antibodies were purchased from BioLegend, eBioscience or Invitrogen.
Anti cd49d
Manufactured by BioLegend
Sourced in United States
Anti-CD49d is a monoclonal antibody that recognizes the CD49d (α4 integrin) antigen. CD49d is a subunit of the VLA-4 (Very Late Antigen-4) integrin complex, which plays a role in cell adhesion and migration.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Monensin, by BioLegend (2 mentions)
Anti foxp3, by BioLegend (1 mentions)
Attune flow cytometer, by Thermo Fisher Scientific (1 mentions)
Anti gr1, by BioLegend (1 mentions)
Anti cd11a, by BioLegend (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Anti cd4, by BioLegend (1 mentions)
Anti cd11b, by BioLegend (1 mentions)
Anti ly6c, by BioLegend (1 mentions)
Anti ly6g, by BioLegend (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Anti cd8, by BioLegend (1 mentions)
Anti ifn γ, by BioLegend (1 mentions)
Soluble anti cd28, by BioLegend (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Golgiplug, by BD (1 mentions)
Anti cd3, by BD (1 mentions)
Staphylococcal enterotoxin b, by Merck Group (1 mentions)
5 protocols using anti cd49d
1
Multiparameter Flow Cytometry Analysis
2
Activation and Stimulation of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryopreserved PBMCs were rapidly thawed, washed twice in pre-warmed RPMI-1640 medium, and rested overnight in complete RPMI-1640 containing 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 10 mM HEPES and 1 mM sodium pyruvate (Invitrogen, Carlsbad, CA). The next day, rested PBMCS were resuspended in fresh media and 1x106 cells/well was seeded into a 96 well plate pre-coated with anti-CD3 (2 µg/mL; BD Biosciences, Catalog No. 550368). Soluble anti-CD28 (1 µg/mL; Biolegend, Catalog No. 302934) and anti-CD49d (1 µg/mL; Biolegend, Catalog No. 304340) was added for co-stimulation and the cells stimulated for 2 days at 37⁰ C in 5% CO2. In select experiments, cells were stimulated with 50 ng/mL of Phorbol-12-myristate 13-acetate (PMA) (Sigma Aldrich, Catalog No. P1585) and 1 µM of Ionomycin (Sigma Aldrich, Catalog No. 19657), and 0.07% Golgi-plug (BD Biosciences, Catalog No. 555029) for 6 hours in fresh complete RPMI media.
3
Activation of VZV-specific T cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMC were thawed and rested at 37°C in RPMI-1640 containing 10% FCS for 2
hours then incubated for 2 hours with sucrose gradient purified inactivated VZV
(Meridian Life Science, ELLEN strain), costimulatory antibodies anti-CD28
(eBioscience; CD28.2 [1 µg/mL]) and anti-CD49d (BioLegend; 9F10 [1
µg/mL]), and anti-CD107a-FITC (H4A3; BD Biosciences). Staphylococcal
enterotoxin B (SEB, Sigma, 1 µg/mL) as a positive control and medium only
as a negative control were utilized. After 2 hours, Brefeldin A (Sigma; 5
µg/mL) and monensin (BioLegend; 2µM) were added. After overnight
stimulation at 37°C, cells were washed with PBS before staining.
hours then incubated for 2 hours with sucrose gradient purified inactivated VZV
(Meridian Life Science, ELLEN strain), costimulatory antibodies anti-CD28
(eBioscience; CD28.2 [1 µg/mL]) and anti-CD49d (BioLegend; 9F10 [1
µg/mL]), and anti-CD107a-FITC (H4A3; BD Biosciences). Staphylococcal
enterotoxin B (SEB, Sigma, 1 µg/mL) as a positive control and medium only
as a negative control were utilized. After 2 hours, Brefeldin A (Sigma; 5
µg/mL) and monensin (BioLegend; 2µM) were added. After overnight
stimulation at 37°C, cells were washed with PBS before staining.
4
Cytokine Profiling of CD8+ T Cell-Depleted PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Crypopreserved Ficoll-Hypaque isolated peripheral blood mononuclear cells (PBMC) were thawed and CD8+ T cell depleted to avoid absorption of cytokines from supernatants by CD8+ T cells in the assay with Dynal CD8+ depletion kits (Life Technologies, Carlsbad, CA) resulting in >90% depletion of CD8+ T cells in PBMC samples (not shown). 0.5 x 106 cells were then plated in duplicate in the bottom chamber of a 96-well HTS transwell plate (3μm pore size) (Corning Inc., Corning, NY) in 100μL of RPMI 1640 medium (Life Technologies) supplemented with 10% FCS, L-glutamine and penicillin/streptomycin (Life Technologies). Duplicate wells were stimulated for 6 days with either 1μg/mL Staphylococcal Enterotoxin B (SEB) (Toxin Technologies, Sarasota, FL); 1μg/mL HCV NS3 antigen (Mikrogen GmbH, Munich, Germany); 1μg/mL HCV NS4 antigen (Mikrogen), 1μg/mL HCV Core antigen (Austral Biologicals, San Ramon, CA) or medium alone. Anti-CD28 (BD Pharmingen, Franklin Lakes, New Jersey) and anti-CD49d (Biolegend, San Diego, CA), (both at 1μg/ml) were added with the antigens for costimulation. Anti-IL-21 and IL-17A antibody coated polycarbonate beads were added to the upper chamber after 72 hours (Millipore Corporation, Billerica, MA). At day 6 of culture, beads in the upper chambers were washed twice and analyzed on a Bioplex Reader (Bio-Rad laboratories, Redmond, WA).
5
Comprehensive PBMC Cytokine Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
About 150,000 peripheral blood mononuclear cells (PBMCs) per well were stimulated in a 96-well plate with peptides (2.5 ng/ml), 1 μg/ml anti-CD28, anti-CD49d, and anti-CD107a FITC (BioLegend) for 6 hr. After 30 min, 5 μg/ml Brefeldin A and 1× Monensin (BioLegend) were added. The positive control contained 2.5 ng/ml PMA and 25 ng/ml ionomycin, and the negative cells and CD28/CD49d. Cells were washed and stained with LIVE/DEAD Fixable Violet stain (Invitrogen), fixed, and permeabilized with BD Fix/Perm solution (BD) and stained with a mix of fluorochrome-conjugated antibodies: CD3 EDC, CD8 PerCP (BD), interleukin (IL)-2 PE, IFN-γ, Pe-Cy7, and tumor necrosis factor (TNF)-α APC (BioLegend). Unstained and singly stained cells were used as controls to calculate compensation. The cell fluorescence intensities were acquired on a Cyan ADP Cytometer (Beckman Coulter) and analyzed with Summit software (Dako).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!