Ex vivo T cell responses were measured based on T cell activation assay previously described by Bacher et al.23 (link) PBMC were thawed and rested overnight, plated at 10 × 106 cells per well in a 6-well plate. The next morning, cells were stimulated with HiMO peptide megapool (2 μg/ml), Phorbol myristate acetate (PMA) and Ionomycin (Io) (positive control) or DMSO (negative control) in the presence of 1 μg/ml anti-CD40 (Miltenyi Biotech, Auburn, CA). Cells were incubated for 6hrs, adding Brefeldin A (1 μg/ml) for the last 3hrs. After the incubation, cells were labeled with anti-CD4 APC ef780, anti-CD3 AF700, anti-CD8/CD14/CD19 V500 and live/dead fixable viability dye (Life technologies, San Diego, CA). After staining and washing, cells were fixed, permeabilized and intracellular staining was performed with anti-CD154 FITC (BD, San Diego, CA). Finally, cells were washed and acquired by flow cytometry using a BD LSR II flow cytometer and data were analyzed using FlowJo software (TreeStar, Ashland, OR). All data acquisition was performed blinded. Gating strategy is shown in Supplemental Fig. S2 .
Anti cd154 fitc
Manufactured by BD
Sourced in United States
Anti-CD154 FITC is a fluorochrome-conjugated monoclonal antibody that binds to the CD154 (CD40 ligand) cell surface protein. It is used in flow cytometry applications for the identification and analysis of CD154-expressing cells.
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Lab products found in correlation
Anti cd40, by Miltenyi Biotec (1 mentions)
Live dead fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Anti cd4 percp cy5, by Thermo Fisher Scientific (1 mentions)
Anti cd4 pe cy7, by BD (1 mentions)
8 color canto 2 flow cytometer, by BD (1 mentions)
Fix perm kit, by Thermo Fisher Scientific (1 mentions)
Anti cd3 v500, by BD (1 mentions)
Anti cd3 apc efluor780, by Thermo Fisher Scientific (1 mentions)
Anti tlr 2 pe, by BD (1 mentions)
Lyse fix buffer, by BD (1 mentions)
2 protocols using anti cd154 fitc
1
Ex vivo T Cell Activation Assay
2
Phenotyping of CD4+ T-cell Subsets
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Fresh whole blood lymphocytes were used for CD4 + T-cell phenotyping without a separation procedure because separation alters chemokine receptor expression. CD4 + T-cell phenotyping has been performed within the 2 h after sampling. We distinguished T-cell subsets according to CD45RA and CCR7 expression: the CM subset (CD45RA -CCR7 + ) and EM subset (CD45RA -CCR7 -) were analyzed. The expression of each HLA-DR, CD40, CD134, CD154, Tbet, and TLR was evaluated in CD4 + T cells and in memory subsets. Leukocytes were labeled with the following antibodies: anti-CD3 V500, anti-CD4 PE-Cy7, anti-CCR7 V450HZ, anti-CD45RA APC or PE-Cy7, anti-CD40 PE-Cy5, anti-CD134 FITC, anti-TLR2 PE (all from BD Biosciences, San Jose, CA, USA), anti-CD4 PerCP-Cy5.5, anti-CD3 APC-eFluor780, and anti-HLA-DR APC-eFluor780 (all from eBioscience, San Diego, CA, USA). RBCs were lysed with Lyse/Fix buffer (BD Biosciences). Cells were fixed and permeabilized using a fix/perm kit (eBioscience) according to manufacturer's instructions. Intracellular markers were detected with anti-CD154 FITC, anti-Tbet AF647 (BD Biosciences), anti-TLR3 FITC, and anti-TLR9 PE (Novus Biologicals, Littleton, CO, USA). Fluorescence was collected on an 8-color Canto II flow cytometer (BD Biosciences).
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