PBMCs were stimulated with either PMA/Iono (Cell stimulation cocktail, eBioscience), plate-bound CD3/CD49d/soluble CD28 (at 10 μg/mL, 5 μg/mL, and 5 μg/mL respectively) or with peptides or their mixtures [Influenza Matrix peptide57–66 (AnaSpec, San Jose, CA), EBV BZLF-1 peptide pool, CMV pp65 peptide pool (both Miltenyi Biotec, San Diego, CA) or HIV-1 PTE Gag peptide pool43 (link) (obtained from the NIH AIDS reagent program)] for 3 hrs in the presence of brefeldin A (eBioscience). All peptides were used according to manufacturer’s instructions at approximately 1 μg/mL of each peptide; peptide mixtures were constructed as sets of 15-mers overlapped by 9 amino acids. Phenotype and cytokine production were evaluated by flow cytometry as described above. In the phospho ERK blocking experiment, MAPK inhibitor U0126 (Tocris Bioscience, Bristol, UK) was added to PBMCs for 3 h prior stimulation with PMA/Iono.
Cmv pp65 peptide pool
Manufactured by Miltenyi Biotec
The CMV pp65 peptide pool is a collection of overlapping peptides derived from the cytomegalovirus (CMV) pp65 antigen. The pool is designed to provide a comprehensive representation of the pp65 protein sequence and can be used for the detection and analysis of CMV-specific T cell responses.
Automatically generated - may contain errors
Lab products found in correlation
Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Influenza matrix peptide57 66, by AnaSpec (2 mentions)
Ebv bzlf 1 peptide pool, by Miltenyi Biotec (2 mentions)
Mapk inhibitor u0126, by Bio-Techne (2 mentions)
Bio plex manager software v 6, by Bio-Rad (1 mentions)
Bio plex pro human cytokine th1 th2, by Bio-Rad (1 mentions)
Magnetic bead, by Miltenyi Biotec (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Gm csf, by R&D Systems (1 mentions)
4 protocols using cmv pp65 peptide pool
1
Multimodal PBMC Stimulation and Analysis
2
Multimodal PBMC Stimulation and Analysis
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PBMCs were stimulated with either PMA/Iono (Cell stimulation cocktail, eBioscience), plate-bound CD3/CD49d/soluble CD28 (at 10 μg/mL, 5 μg/mL, and 5 μg/mL respectively) or with peptides or their mixtures [Influenza Matrix peptide57–66 (AnaSpec, San Jose, CA), EBV BZLF-1 peptide pool, CMV pp65 peptide pool (both Miltenyi Biotec, San Diego, CA) or HIV-1 PTE Gag peptide pool43 (link) (obtained from the NIH AIDS reagent program)] for 3 hrs in the presence of brefeldin A (eBioscience). All peptides were used according to manufacturer’s instructions at approximately 1 μg/mL of each peptide; peptide mixtures were constructed as sets of 15-mers overlapped by 9 amino acids. Phenotype and cytokine production were evaluated by flow cytometry as described above. In the phospho ERK blocking experiment, MAPK inhibitor U0126 (Tocris Bioscience, Bristol, UK) was added to PBMCs for 3 h prior stimulation with PMA/Iono.
3
Cytokine Profiling of CMV-Stimulated Cells
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Cryopreserved expanded cells were stimulated with CMV pp65 peptide pool (Miltenyi) 50 ng/mL at a cell concentration of 1E+06 cells/mL in a 96-well plate. Supernatant samples were collected at 0, 6, 10, 24, and 48 h. All samples were centrifuged at 1,000 g for 15 min at 4°C. To remove aggregates or debris, samples were centrifuged again at 10,000 g for 10 min at 4°C and stored at −80°C until analysis. Samples were thawed just once and kept on ice before assay. The Bio-Plex Pro™ Human Cytokine Th1/Th2 (Bio-Rad Laboratories, Hercules, CA, USA) was used for the determination of levels of 9 cytokines: IL-2, IL-4, IL-5, IL-10, IL-12 (p70), IL-13, IFN-γ, GM-CSF, and TNF-α in a multiplex assay using a Luminex 100IS analyzer (Luminex Corp. Austin. TX, USA) according to the manufacturer's instructions. Duplicates were tested for each sample. Data analysis was performed using Bioplex Manager Software v6.1 (Bio-Rad Laboratories Inc.).
4
T-cell Proliferation and Cytokine Profiling
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Cell proliferation was measured using the carboxy fluoroscein succinimidyl ester (CFSE) dilution assay [87 (link)], following co-culture with antigen-loaded autologous monocyte-derived dendritic cells (MDDC). Briefly, monocytes isolated from PBMCs by negative selection using magnetic beads (Miltenyi Biotec) [86 (link)] were differentiated into MDDC in the presence of GM-CSF and IL-4 (20 ng/ml; R&D Systems). MDDCs were loaded with SEB (25 ng/ml; Toxin Technologies), CMV-pp65 peptide pool (1 μg/ml; Miltenyi) or Candida albicans hyphae LAM-1 [88 (link)] (25 µl protein lysate [87 (link)]) for one hour at 37 °C and cocultured with FACS-sorted CFSE-loaded T-cell subsets. MDDC:T-cell cocultures (1:4 ratio) were maintained for 5 days at 37 °C. MDDC:T-cell co-cultures were stained with CD3 (T-cell marker) and CD1c (DC marker). Proliferating T-cells were identified as cells with a CFSElowCD3+CD1c− phenotype. In parallel, MDDC:T-cell co-cultures were stimulated with PMA and Ionomycin in the presence of brefeldin A and the expression of cytokines in proliferating T-cell subsets was quantified by FACS upon intracellular staining with specific anti-cytokine Abs (See table above). Intracellular expression of cytokines was quantified by FACS [87 (link)].
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