Human kidney cells 293 (ATCC® CRL-1573) were cultured in high glucose Dulbecco’s Modified Eagles Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA). HepG2-NTCP cells (kindly provided by Prof. Ulrike Protzer) were cultured in DMEM with 10% FBS as well. Cryopreserved primary human hepatocytes (PHHs) obtained from BD Biosciences (Woburn, MA, USA) were cultured in William’s E Medium (WEM, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS, 1% Penicillin/Streptomycin, 0.17 μM of human insulin (Sigma Aldrich, St. Louis, MO, USA), 10 μM of hydrocortisone 21-hemisuccinate (Sigma Aldrich, St. Louis, MO, USA), and 1.8% DMSO. HepG2-NTCP cells and PHHs were transduced by AdVs at a multiplicity of infection (MOI) of 1. The eBioscience™ protein transport inhibitor cocktail (PTI, Thermo Fisher Scientific, Waltham, MA, USA) and Thapsigargin (Tg, Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls for induction of ER stress and apoptosis, respectively.
Ebioscience protein transport inhibitor cocktail
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EBioscience Protein Transport Inhibitor Cocktail is a ready-to-use solution designed to inhibit the cellular transport of proteins. It is used to prevent the secretion or release of cytokines, chemokines, and other proteins from stimulated cells during in vitro cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ebioscience cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Human insulin, by Merck Group (1 mentions)
Hydrocortisone 21 hemisuccinate, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Cd8 clone rpa t8, by BD (1 mentions)
Anti cd3 clone sk7, by BD (1 mentions)
Fixable viability stain 510, by BD (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Granzyme b clone gb11, by BD (1 mentions)
Cd4 clone rpa t4, by BD (1 mentions)
Anti human cd28 clone cd28, by BD (1 mentions)
Anti cd49d clone 9f10, by BioLegend (1 mentions)
Ifn γ clone b27, by BD (1 mentions)
Polymyxin b, by Merck Group (1 mentions)
8 protocols using ebioscience protein transport inhibitor cocktail
1
Cultivating Human Cell Lines for ER Stress Study
2
Cytotoxic T-cell Activation Assay
3
Peptide-Loaded Nanoparticle Stimulation of T Cells
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E+E cells were prepared in fresh TexMACS the day after thawing and 1.0x105 cells/well were seeded into a 96-well round-bottom plate (Corning, Corning, NY). Either peptide loaded nanoparticles or phorbol 12-myristate 13-acetate/Ionomycin in Invitrogen’s Cell Stimulation Cocktail (plus protein transport inhibitors, 500X) (Thermo Fisher Scientific) were used to stimulate cells. Cells alone with Invitrogen’s eBioscience Protein Transport Inhibitor Cocktail (Thermo Fisher Scientific) were used as the unstimulated control and when stimulating cells with peptide loaded nanoparticles. Cells were fixed, permeabilized, and stained before analyzing as described above.
4
Intracellular Protein Analysis in PBMC-Organoid Co-Cultures
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Five hours before collection, cultures were treated with eBioscience Protein Transport Inhibitor Cocktail (500X, ThermoFisher) to facilitate intracellular accumulation of temporally expressed soluble proteins. Duplicate wells of PBMC–organoid co-cultures from each condition were collected at 5, 24, 48 and 72 h post treatment. Co-cultures were first washed with PBS and then digested to single cells using Accutase solution. Cell suspensions were passed through a 70 µm strainer and stained for surface proteins. Cells were then fixed and permeabilized using the eBioscience Foxp3 Transcription Factor Staining Buffer set (ThermoFisher) and subsequently stained for intracellular and intranuclear proteins (Supplementary Table 2 ). Stained cell suspensions were acquired on a BD Fortessa X-20 flow cytometer (BD Biosciences) and analysed using FlowJo v.10.
5
Induction and Characterization of Murine T Helper and Cytotoxic T Cells
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All cells were cultured in RPMI 1640 supplemented with 10% FCS, 4 mM GlutaMAX, 20 mM Hepes, 0.05 mM 2-mercaptoethanol, penicillin (1000 U/ml), and streptomycin (1 mg/ml) (all from Thermo Fisher Scientific). Purified CD4+ T cells or CD8+ T cells were stimulated with Dynabeads mouse T-activator CD3/CD28 (Thermo Fisher Scientific) at a bead-to-cell ratio of 2:1 for 4 days in the presence of recombinant murine IL-7 (10 ng/ml) (PeproTech) for TH1 or Tc1 cell induction in 24-well plates (Corning). To detect intracellular cytokines, cells were restimulated with eBioscience Cell Stimulation Cocktail (Thermo Fisher Scientific) for 5 hours plus eBioscience Protein Transport Inhibitor Cocktail (Thermo Fisher Scientific). To detect IFN-γ and IL-2 production, T cells were stimulated with Dynabeads mouse T-activator CD3/CD28 for 2 days. The production of IFN-γ or IL-2 was measured by an enzyme-linked immunosorbent assay kit (BioLegend).
6
Spike Protein-Specific T Cell Activation Assay
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Cryopreserved PBMCs were thawed and rested overnight. Cells were then stimulated with a combined spike S1 and S2 peptide pool, at a final concentration of 1 μg ml−1 per peptide or DMSO (mock). After 1 h, eBioscience protein transport inhibitor cocktail (Thermo Fisher Scientific) was added and cells were incubated for a further 5 h. eBioscience cell stimulation cocktail (Thermo Fisher Scientific) was used as a positive control. After stimulation, cells were washed (PBS + 0.1% BSA) and surface-stained at 4 °C for 30 min. Cells were then washed and fixed in 2% paraformaldehyde. After washing, brilliant staining buffer (BD Bioscience) and a final concentration of 0.4% saponin were added. Cells were stained intracellularly at room temperature for 30 min. Cells were then washed and run on a BD FACSymphony A3 flow cytometer (BD Biosciences). Antibody details are provided in Supplementary Table 4 .
7
Stimulation and Analysis of Splenocytes
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Isolated splenocytes or PLN-derived cells were cultured in a 96-well round bottom plate (Corning), stimulated with 0–10 μg/mL sBDC (Anaspec) or IGRP206–214 (Anaspec), pelleted, and then incubated at 37°C for 30–60 minutes for subsequent phospho- or ImageStream flow cytometry. In some experiments, cells were cultured in a round-bottom 96-well plate and stimulated 4 hours with PMA (1:2000; Sigma-Aldrich) and ionomyocin (1:1000; Sigma-Aldrich) or 1 μg/mL sBDC (Anaspec) in the presence of brefelden A (1:1,000; BioLegend), monensin (1:1,000; BioLegend), and eBioscience protein transport inhibitor cocktail (1:500; Invitrogen).
8
Multiparametric Analysis of Splenocyte Responses
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Two million splenocytes were cultured for 5–6 hours with the peptide pools used above, as previously described [44 (link)], and with eBioscience protein transport inhibitor cocktail (Invitrogen). Surface (for CD4 and CD8) and intracellular (for remaining markers) staining followed. Biolegend anti-mouse antibodies conjugated to fluorophores used in this experiment included CD3ε-PE/Cy5 (145-2C11), CD4-FITC (RM4-5), CD8a-APC/Cy7 (53-6.7), IFNγ-APC (XMG1.2), TNFα-BV605 (MP6-XT22), and IL-2-PE-Cy7 (JES6-5H4). Live-dead exclusion was performed using violet fluorescent reactive dye (Invitrogen). Data was collected using a BD Biosciences LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo v10 (FlowJo LLC, Ashland, OR, USA).
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