PBMCs were thawed and washed, and CD8+ T cells were depleted using human CD8 MicroBeads (Miltenyi Biotec, 130-045-201). CD8+ T cell–depleted PBMCs were cultured in 24-well plates at a concentration of 10 × 106 cells/ml in RPMI 1640 supplemented with Hepes, penicillin/streptomycin, and 10% human serum (Sigma-Aldrich). Cells were rested for 3 h and then stimulated with 0.5 µg/ml of HIV-1 consensus B Gag pool, CMV peptide pool, and CEFT peptide pool (PM-HIV-CONB, PM-PP65-2, PM-CEFT-MHC-II, all from JPT Peptide Technologies) for 18 h at 37°C, 5% CO2. A MOG peptide pool (0.5 µg/ml, PM-MOG, JPT Peptide Technologies) served as negative control. SEB-stimulated cells (0.5 µg/ml, Toxin Technology, BT202) served as positive control. Cells were stained for viability with Aquavivid (Life Technologies) at 1/200 in PBS for 20 min, 4°C; incubated with human FcR Block (Miltenyi Biotec, 130-059-901) at 1/70 in PBS-FBS 1% for 10 min, 4°C; and stained with antibodies to surface markers for 30 min, 4°C with Brilliant Violet Buffer (BD Biosciences, 566349) at 1/4 in PBS-FBS 1% (see Table S4 for antibody staining panel).
Pm pp65 2
Manufactured by JPT Peptide Technologies
The PM-PP65-2 is a laboratory instrument designed for peptide synthesis. It is a programmable microwave peptide synthesizer that can be used for the automated synthesis of peptides. The device features a microwave irradiation system that can accelerate the synthesis process. The core function of the PM-PP65-2 is to facilitate the automated production of peptides in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Brilliant violet buffer, by BD (2 mentions)
Human serum, by Merck Group (2 mentions)
Human cd8 microbeads, by Miltenyi Biotec (2 mentions)
Aquavivid, by Thermo Fisher Scientific (2 mentions)
Human fcr block, by Miltenyi Biotec (2 mentions)
Pm hiv conb, by JPT Peptide Technologies (2 mentions)
Pm ceft mhc 2, by JPT Peptide Technologies (2 mentions)
Pm mog, by JPT Peptide Technologies (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Bt202, by Toxin Technology (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
2 protocols using pm pp65 2
1
Stimulation and Characterization of PBMCs
2
Antigen-specific T Cell Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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PBMCs were thawed, washed, and CD8 + T cells were depleted using human CD8 MicroBeads (Miltenyi, . CD8 + T cell-depleted PBMCs were cultured in 24 well plates at a concentration of 10 x 10 6 cells/ml in RPMI 1640 supplemented with HEPES, penicillin/streptomycin and 10% human serum (Sigma). Cells were rested for 3h and then stimulated with 0.5 ug/ml of HIV-1 consensus B Gag pool, CMV peptide pool, and CEFT peptide pool (PM-HIV-CONB, PM-PP65-2, PM-CEFT-MHC-II, all from JPT Peptide Technologies) for 18h at 37 o C, 5% CO2. A MOG peptide pool (0.5 ug/ml, PM-MOG, JPT Peptide Technologies) served as negative control. SEB-stimulated cells (0.5 ug/ml, Toxin technology, Cat# BT202) served as positive control. Cells were stained for viability with Aquavivid (Life Technologies) at 1/200 in PBS for 20 min, 4 o C; incubated with human FcR Block (Miltenyi Biotec, Cat#130-059-901) at 1/70 in PBS-FBS 1% for 10 min, 4 o C; and stained with antibodies to surface markers for 30 min, 4 o C with Brilliant Violet Buffer (BD Biosciences, Cat# 566349) at 1/4 in PBS-FBS 1% (see Supplemental Table 3 for antibody staining panel).
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