Human PBMCs were isolated from the peripheral blood of healthy donors by standard density gradient centrifugation on Ficoll-Hypaque (GE Healthcare-Life Sciences, USA). Regulatory T cells (Tregs) were isolated from PBMCs using magnetic beads technology (CD4+ CD25+ CD127dim/- Regulatory T Cell Isolation Kit II; Miltenyi Biotec, USA) according to the manufacturer’s recommendations. Autologous PBMCs were stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 and 0.5 µg/mL respectively; BD Biosciences, USA) in the presence of Tregs (1:1 PBMC to Treg ratio). The therapeutic antibodies were added, and after 3 days of culture, cytokine levels were measured in the culture supernatant using ProcartaPlex immunoassay according to the manufacturer’s recommendations (Th1/Th2/Th9/Th17/Th22/Treg Cytokine 18-Plex Human ProcartaPlex™ Panel; Thermo Fisher Scientific). Results were expressed as a percentage of inhibition of cytokine secretion relative to the level secreted in the absence of Tregs.
18 plex human procartaplex panel
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 18-Plex Human ProcartaPlex™ Panel is a multiplex immunoassay panel designed to detect and measure the concentration of 18 different human protein analytes simultaneously in a single sample. The panel utilizes magnetic bead-based technology and is compatible with the Luminex® platform.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll hypaque, by GE Healthcare (2 mentions)
Cd4 cd25 cd127dim regulatory t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Soluble anti cd3 and anti cd28 antibodies, by BD (1 mentions)
Ifnγ elisa, by R&D Systems (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Memory cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Magnisort human cd14 positive selection kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using 18 plex human procartaplex panel
1
Treg-mediated Inhibition of Cytokine Secretion
2
Paracrine Factors in Co-Culture Conditioned Media
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The levels of different paracrine factors involved were determined in each co-culture-conditioned medium compared to conditioned media secreted by controls (hA-MSCs grown alone in RPMI and A-WBCs grown alone in AF) in triplicate. Cytokine analysis was completed using a Th1/Th2/Th9/Th17/Th22/Treg Cytokine 18-Plex Human ProcartaPlex Panel (Thermo Fisher Scientific, Waltham, MA, USA) based on Luminex technology, according to the manufacturer’s instructions. The data were acquired with xPONENT® 3.1 software for Luminex 100/200 (Luminex Corporation, Austin, TX, USA). The concentration of each factor was calculated via interpolation from standard curves. Results are shown as fold increases of each conditioned medium. ProcartaPlex™ Analyst 1.0 was used for analyzing the data obtained.
3
Antibody Modulation of Cytokine Secretion
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Blood from healthy human donors or cynomolgus monkey (COVANCE) was collected in heparin tubes and processed the same day. Briefly, blood was diluted tenfold with AIM-V® Medium (ThermoFisher Scientific) and stimulated with 5 ng/mL staphylococcus enterotoxin B (SEB; Toxin Technology, USA). Various concentrations of therapeutic antibodies were added, and after 3 days of culture, cytokine levels were assessed in the culture supernatant. Cytokine levels in human samples were determined using ProcartaPlex immunoassay (Th1/Th2/Th9/Th17/Th22/Treg Cytokine 18-Plex Human ProcartaPlex™ Panel; Thermo Fisher Scientific) and cynomolgus monkey samples by IFNγ ELISA (R&D Systems) according to the manufacturer’s recommendations. Results were expressed as the fold change in cytokine secretion relative to the level secreted in the absence of therapeutic antibodies.
4
Therapeutic Antibody Effects on Viral-specific T Cells
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Human PBMCs were isolated from the peripheral blood of healthy donors by standard density gradient centrifugation on Ficoll-Hypaque (GE Healthcare-Life Sciences, USA). Memory CD4+ T cells were enriched by magnetic beads separation (Memory CD4+ T Cell Isolation Kit; Miltenyi Biotec, USA) according to the manufacturer’s recommendations. Autologous monocytes were isolated by magnetic beads separation (MagniSort™ Human CD14 Positive Selection Kit; Thermo Fisher Scientific, USA) according to the manufacturer’s recommendations. Autologous memory CD4+ T cells and monocytes were mixed at a 1:1 ratio, and viral-specific T cells were re-stimulated through the addition of a pool of selected peptides from cytomegalovirus, Epstein–Barr virus, influenza and tetanus toxin (CEFT) (0.1 µg/mL of each peptide; Axxora, USA). Various concentrations of therapeutic antibodies were added, and after 7 days of culture, cytokine levels were assessed in the culture supernatant using ProcartaPlex immunoassay according to the manufacturer’s recommendations (Th1/Th2/Th9/Th17/Th22/Treg Cytokine 18-Plex Human ProcartaPlex™ Panel; Thermo Fisher Scientific). Results were expressed as the fold change in cytokine secretion relative to the level secreted in the absence of therapeutic antibodies.
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