T2 cells were incubated with 100 µM of each peptide in R0 supplemented with 100 ng/ml human β2m at 37°C for 16 h, as previously described (16 (link)). The cells were then washed four times to remove free peptides, incubated with 0.2% GoldiPlug (BD Biosciences Pharmingen, san Diego, CA, USA) in R0 for 1 h to block the cell surface expression of newly synthesized HLA-A2.1. After washing and incubation for 0, 2, 4, 6 or 8 h, the cells were stained with anti-HLA-A2.1 antibody (BB7.2). For each time-point, the peptide-induced HLA-A2.1 expression was calculated as: mean fluorescence of peptide-preincubated T2 cells - mean fluorescence of T2 cells treated in similar conditions in the absence of peptide. DC50 (dissociation complex, DC) was defined as the time required for the loss of 50% of the HLA-A2.1/peptide complexes stabilized at t=0.
Goldiplug
Manufactured by BD
Sourced in United States
GoldiPlug is a laboratory equipment product designed to provide a secure and reliable connection for various laboratory applications. It is a device that creates a tight seal, preventing leaks and ensuring the integrity of the system. The core function of GoldiPlug is to facilitate the connection and sealing of laboratory equipment, enabling efficient and controlled fluid or gas transfer.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Ionomycin calcium salt, by Merck Group (1 mentions)
Fixation permeabilization buffer, by BD (1 mentions)
Fixation permeabilization kit, by BD (1 mentions)
Fitc conjugated mouse anti human cd4, by BD (1 mentions)
40 m cell strainer, by BD (1 mentions)
Fortessa cell analyzer, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
4 protocols using goldiplug
1
Peptide-induced Stabilization of HLA-A2.1
2
Intracellular Cytokine Staining Protocol
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Intracellular staining, for IFN- γ, Birc5 and Foxp3, was performed with antibodies and fixation/permeabilization buffers (BD Biosciences) or Foxp3/transcription factor staining buffer set (Thermo Fisher). For IFN- γ staining, the cells were stained after treatment with phorbol 12-myristate 13 acetate (Sigma-Aldrich, St. Louis, MO, USA), ionomycin calcium salt (Sigma-Aldrich), and Goldiplug (BD Biosciences).
3
WT1-specific helper T cell induction
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The induction of WT1-specific helper T cells was also measured. Human PBMCs positive for HLA-DRB1*14:54/15:01, HLA-DQB1*05:03/06:03, and HLA-DPB1*04:01/04:02were incubated for 10 days with adegramotide (Bachem). On Day 10, 40 μg/mL adegramotide or 0.125% DMSO were added and incubated for 3.5 h. Following an overnight incubation with GoldiPlug (BD Biosciences), cells were stained for CD4 (FITC-conjugated mouse anti-human CD4; BD Biosciences), fixed using a Fixation/Permeabilization Kit (BD Biosciences), and stained with phycoerythrin-conjugated mouse anti-human interferon-gamma (IFN-γ) (BD Biosciences).
4
Quantifying CTL Activation by Nanogel Vaccines
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CTL activation by the nanogel vaccines was evaluated as follows26 (link). The spleen was harvested from an immunized mouse and the cell suspension was re-suspended in RPMI 1640. After ammonium-chloride-potassium buffer treatment to lyse the red blood cells, the remaining cells were filtered through a 40-µm cell strainer (BD Biosciences). The cells were incubated with the H-2Kb OVA epitope peptide at 37 °C for 1 h, and then with Goldi Plug (BD Biosciences) overnight. The collected cells were stained with fluorescent dye-labelled anti-CD8a or IFN-γ antibodies at 4 °C for 20 min, and then fixed using the Cytofix/Cytoperm kit (BD Biosciences). The fluorescence intensities were measured by LSR (Fortessa cell analyzer, BD Biosciences) and the data obtained were analysed using FlowJo 7.6.5 software (Tree Star).
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