Cells were thawed and plated at concentrations of 0.5 to 1 × 106 in 200 µL of Roswell Park Memorial Institute (RPMI) growth medium 1640 (Gibco) supplemented with 1% sodium pyruvate (NEAA), 100 U/mL penicillin/streptomycin (Gibco), and 10% fetal bovine serum at 37 °C and 5% CO2. PBMCs were then stimulated with S peptide pools in culture over the course of 7 d. On day 1, cells were washed and stimulated with PepMix SARS-CoV-2 S glycoprotein pool 1 and pool 2 (GenScript) at a final concentration of 1 µg/mL per peptide; stimulation controls received PBS. Cells were incubated for 6 d with a single media change performed on day 4. On day 6, cells were restimulated with 10 µg/mL per peptide for 12 h, with the last 6-h incubation performed in the presence of the protein transport inhibitor mixture (ThermoFisher; 1:500 dilution). Following this incubation, cells were washed with PBS 2 mM ethylenediaminetetraacetic acid and prepared for analysis by flow cytometry.
Protein transport inhibitor mixture
Manufactured by Thermo Fisher Scientific
Protein transport inhibitor mixture is a solution designed to disrupt the intracellular transport of proteins within cells. The mixture contains a combination of chemical compounds that interfere with various cellular processes involved in protein trafficking and localization. This product is intended for use in research applications to investigate protein transport mechanisms.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ifnγ apc cy7, by Thermo Fisher Scientific (1 mentions)
Cd3 alexa fluor 700, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc, by BioLegend (1 mentions)
Intracellular fixation and permeabilization buffer set, by Thermo Fisher Scientific (1 mentions)
Ifnγ apc cy7, by BioLegend (1 mentions)
Rat anti mouse cd16 cd32 monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Zombie red, by BioLegend (1 mentions)
Cell stimulation mixture, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
4 protocols using protein transport inhibitor mixture
1
SARS-CoV-2 Spike Glycoprotein Peptide Stimulation
2
Measuring Antigen-Specific T Cell Cytokines
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The preparation of single-cell spleens was carried out as previously described [25 (link)]. 7 days after the boost immunization, splenocytes were stimulated with S. Typhimurium LPS (50 ng/µL) for 8 h at 37 °C with 5% CO2 in the presence of a protein transport inhibitor mixture (Thermo Fisher). Cytokine expression of CD4+ T cells was measured using rat anti-mouse CD3-Alexa Fluor 700, CD4-FITC, IFNγ-APC/Cy7, and IL-4-Alexa Fluor 647 (eBioscience). The ratio of cytokine-positive cells in the unstimulated sample was subtracted from the antigen-stimulated value of the same mouse to calculate the percentage of antigen-specific cells.
3
Multicolor Flow Cytometry Analysis of T Cell Cytokines
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Single-cell preparations of spleen were obtained as previously described (47 (link)). Splenocytes were stimulated with each type of the CPSs or with the LPS of χ3761 (50 ng/2 × 106 cells) in the presence of Protein Transport Inhibitor Mixture (eBioscience). The cells were then incubated for 8 h at 37 °C with 5% CO2. Before staining, dead cells were excluded using Zombie Red (Biolegend) and mouse FcR were blocked with the rat anti-mouse CD16/CD32 monoclonal antibody (eBioscience). For surface staining, cells were incubated with fluorophore-conjugated antibodies on ice for 30 min. The eBioscience Intracellular Fixation and Permeabilization Buffer Set was used for intracellular staining. Cells were run on a BD LSRFortessa cell analyzer and analyzed with FlowJo software (TreeStar).
For detection of cytokine expression in CD4+ T cells at 21 d after primary immunization, the following antibodies purchased from Biolegend were used, including rat anti-mouse CD3-Alexa Fluor 700, CD4-FITC, IFN-γ–APC/Cy7, APC/Cy7-conjugated rat IgG1κ (isotype), IL-4–Alexa Fluor 647, Alexa Fluor 647-conjugated rat IgG1κ (isotype). Data are presented as described in the figure legends.
For detection of cytokine expression in CD4+ T cells at 21 d after primary immunization, the following antibodies purchased from Biolegend were used, including rat anti-mouse CD3-Alexa Fluor 700, CD4-FITC, IFN-γ–APC/Cy7, APC/Cy7-conjugated rat IgG1κ (isotype), IL-4–Alexa Fluor 647, Alexa Fluor 647-conjugated rat IgG1κ (isotype). Data are presented as described in the figure legends.
4
Cytokine Profile of Tumor-Infiltrating T Cells
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The tumor single-cell suspension was prepared as above described. For antigen-specific stimulation, the cells were plated in 24-well plates and pulsed with TRP2180–188 (SVYDFFVWL) for 16 h at 37 °C in complete T cell media (RPMI 1640, 10% FBS, 50 μM β-mercaptoethanol, 100 U/mL Pen/Strep, 1× MEM Non Essential Amino Acids Solution, 1 mM sodium pyruvate) with ebioscience Protein Transport Inhibitor Mixture. For broad stimulation, the cells were plated in 24-well plates in complete T cell media with ebioscience Cell Stimulation Mixture (plus protein transport inhibitors) for 5 h. After stimulation, the cells were collected, washed, blocked with Fc blocker, stained with LIVE/DEAD Fixable Aqua Dead Cell Stain and surface markers (CD45, CD3, TCRb, CD4, CD8), and then fixed using the ebioscience Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer’s instructions. Cells were then washed and permeabilized for intracellular staining for IFN-γ, TNF-α, Foxp3, and IL-17. Then, cells were analyzed on a BD LSRII flow cytometer.
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