Naive precursors specific for HLA-A2–EV10 were primed in vitro using an accelerated dendritic cell (DC) coculture protocol as described previously (11 (link), 16 (link), 17 ). Briefly, thawed PBMCs were resuspended at 5 × 106 cells/well in AIM-V medium (Thermo Fisher Scientific) supplemented with Flt3 ligand (Flt3L; 50 ng/mL; R&D Systems) in the absence or presence of rosiglitazone (40 μM; Sigma-Aldrich) or IL-7 (20 ng/mL; R&D Systems). After 24 hr (day 1), the Melan-A peptide EV20 (1 μM) was added to the cultures, and DC maturation was induced using a standard cocktail of inflammatory cytokines, incorporating IL-1β (10 ng/mL), IL-7 (0.5 ng/mL), PGE2 (1 μM), and TNF (1,000 U/mL) (all from R&D Systems), or ssRNA40 (TLR8L; 0.5 μg/mL; InvivoGen). The cultures were supplemented on day 2 with FCS (10% v/v; Sigma-Aldrich). Medium was replaced every 3 days thereafter with fresh R+ containing FCS (10% v/v; Sigma-Aldrich). Antigen-specific CD8+ T cells were characterized via flow cytometry on day 10.
Ssrna40 tlr8l
Manufactured by InvivoGen
SsRNA40 (TLR8L) is a synthetic single-stranded RNA oligonucleotide that functions as a Toll-like receptor 8 (TLR8) ligand. It is designed to specifically activate TLR8 signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 7 (il 7), by R&D Systems (4 mentions)
Tumor necrosis factor (tnf), by R&D Systems (4 mentions)
Fetal calf serum (fcs), by Merck Group (3 mentions)
Aim 5 medium, by Thermo Fisher Scientific (3 mentions)
Rosiglitazone, by Merck Group (3 mentions)
Flt3 ligand flt3l, by R&D Systems (3 mentions)
Il 1β, by R&D Systems (3 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Flt3l, by R&D Systems (1 mentions)
Prostaglandin e2 pge2, by R&D Systems (1 mentions)
4 protocols using ssrna40 tlr8l
1
Priming Naive HLA-A2–EV10 Specific T Cells
2
Priming Naive HLA-A2–EV10 Specific T Cells
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Naive precursors specific for HLA-A2–EV10 were primed in vitro using an accelerated dendritic cell (DC) coculture protocol as described previously (11 (link), 16 (link), 17 ). Briefly, thawed PBMCs were resuspended at 5 × 106 cells/well in AIM-V medium (Thermo Fisher Scientific) supplemented with Flt3 ligand (Flt3L; 50 ng/mL; R&D Systems) in the absence or presence of rosiglitazone (40 μM; Sigma-Aldrich) or IL-7 (20 ng/mL; R&D Systems). After 24 hr (day 1), the Melan-A peptide EV20 (1 μM) was added to the cultures, and DC maturation was induced using a standard cocktail of inflammatory cytokines, incorporating IL-1β (10 ng/mL), IL-7 (0.5 ng/mL), PGE2 (1 μM), and TNF (1,000 U/mL) (all from R&D Systems), or ssRNA40 (TLR8L; 0.5 μg/mL; InvivoGen). The cultures were supplemented on day 2 with FCS (10% v/v; Sigma-Aldrich). Medium was replaced every 3 days thereafter with fresh R+ containing FCS (10% v/v; Sigma-Aldrich). Antigen-specific CD8+ T cells were characterized via flow cytometry on day 10.
3
In Vitro Priming Assay for Antigen-Specific CD8+ T Cells
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The in vitro priming assay was performed as described previously [27] (link). Briefly, PBMCs (Peripheral Blood Mononuclear Cells) from healthy HLA-A2+ individuals were supplemented with FLT3L (FMS-like tyrosine kinase 3 ligand; 50 ng/ml; R&D Systems) and cultured at 5 × 106 cells/well in a 24-well plate. After 24 h, maturation of DCs was induced with different ligands (at individual concentrations of 10 μM or 10 µM of each for the combination) or a standard cocktail of inflammatory cytokines comprising TNF (1000 U/mL), IL-1β (10 ng/mL), IL-7 (0.5 ng/mL) and prostaglandin E2 (PGE2; 1 μM) (R&D Systems) or the ssRNA40 TLR8L (0.5 μg/mL; Invivogen), added together with the ELA-20 peptide (YTAAEELAGIGILTV ILGVL; Melan-A/MART-1 residues 21–40A27L) at a final concentration of 10 µM. ELA-specific CD8+T cell frequency and phenotype were determined on day 10. Data were acquired using an LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star). The experiments were repeated with 5 donors.
4
Accelerated Dendritic Cell Priming for HLA-A2-EV10
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Naive precursors specific for HLA-A2-EV10 were primed in vitro using an accelerated dendritic cell (DC) coculture protocol as described previously [9, 14, 15] . Briefly, thawed PBMCs were resuspended at 5 × 10 6 cells/well in AIM-V medium (Thermo Fisher Scientific) supplemented with Flt3 ligand (Flt3L; 50 ng/ml; R&D Systems) in the absence or presence of rosiglitazone ( 40μM; Sigma-Aldrich) or IL-7 (20 ng/mL; R&D Systems). After 24 hr (day 1), the Melan-A peptide EV20 (1 µM) was added to the cultures, and DC maturation was induced using a standard cocktail of inflammatory cytokines, incorporating IL-1β (10 ng/mL), IL-7 (0.5 ng/mL), prostaglandin E2 (PGE2; 1 μM), and TNF (1,000 U/mL) (all from R&D Systems), or ssRNA40 (TLR8L; 0.5 μg/mL; InvivoGen). The cultures were supplemented on day 2 with FCS (10% v/v; Sigma-Aldrich). Medium was replaced every 3 days thereafter with fresh R+ containing FCS (10% v/v; Sigma-Aldrich). Antigen-specific CD8 + T cells were characterized via flow cytometry on day 10.
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