Protein transport inhibitor cocktail

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Protein Transport Inhibitor Cocktail is a laboratory product designed to inhibit the transport of proteins. It is intended for use in cell-based assays and experiments where the regulation of protein trafficking is of interest.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Protein transport inhibitor (63 citations) Protein inhibitor cocktail (33 citations) Protein inhibitor (8 citations) Cell stimulation cocktail with protein transport inhibitors (3 citations) Cocktail inhibitor (2 citations) Cell Stimulation Cocktail plus Protein Transport Inhibitors reagent (2 citations)

117 protocols using protein transport inhibitor cocktail

NK Cell Response to SARS-CoV-2 Modulation

Check if the same lab product or an alternative is used in the 5 most similar protocols

NK cells were treated with LLT1 protein (Recombinant Human OCIL/CLEC2d Fc Chimera Protein, CF, R&D Systems, Minneapolis, MN, USA) at 5 µg/ml for 2h or left untreated. Next, NK cells were collected for CD161 surface flow cytometry analysis or were stimulated with a 1×cocktail of PMA and ionomycin (Cell Stimulation Cocktail, eBioscience) for 18h and then added to A549^ACE2/TMPRSS2 cells 24h p.i. with the SARS-CoV-2 or, in the presence of 1×cocktail of Brefeldin A and Monensin (Protein Transport Inhibitor Cocktail, eBioscience), for intracellular analysis of intracellular proteins by flow cytometry, as described above.
CD161 receptor blocking on NK cells was performed by addition of 10 mg/ml of purified CD161-blocking mAb (HP-3G10, BioLegend) or isotype control IgG1 (BioLegend) followed by 2h incubation at 37°C under 5% CO₂. Then, LLT1 protein solution at 5 µg/ml for 2h was added. Then, cytotoxicity assay or degranulation assay was performed, ot the cells were stimulated with a 1×cocktail of PMA and ionomycin (Cell Stimulation Cocktail, eBioscience) for 18h in the presence of 1×cocktail of Brefeldin A and Monensin (Protein Transport Inhibitor Cocktail, eBioscience), for intracellular analysis of intracellular proteins by flow cytometry, as described above.

Lenart M., Górecka M., Bochenek M., Barreto-Duran E., Szczepański A., Gałuszka-Bulaga A., Mazur-Panasiuk N., Węglarczyk K., Siwiec-Koźlik A., Korkosz M., Łabaj P.P., Baj-Krzyworzeka M., Siedlar M, & Pyrc K. (2023). SARS-CoV-2 infection impairs NK cell functions via activation of the LLT1-CD161 axis. Frontiers in Immunology, 14, 1123155.

+ Open protocol

+ Expand

NK Cell Degranulation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

TCD splenocytes (10⁵ cells/200 μl/well) were plated with YAC-1 cells at the indicated ratios in RPMI-1640, 10% HI-FBS, 1,000 IU/ml rhIL-2. V450 anti-CD107a (BD, clone 1D4B, 0.25 μg/well) was added and incubated at 37°C for 1h. Protein transport inhibitor cocktail (ThermoFisher) was then added to each well and further incubated for 4 h. Cells were then washed twice with PBS/2% FBS and stained for CD3 and CD49b. Dead cells were stained with ZNIR (BioLegend). Controls included effector-only cells (non-stimulated) and cells stimulated with PMA+ionomycin (Cell Stimulation Cocktail, Thermo Fisher).

Barisone G.A., O’Donnell R.T., Ma Y., Abuhay M.W., Lundeberg K., Gowda S, & Tuscano J.M. (2018). A purified, fermented, extract of Triticum aestivum has lymphomacidal activity mediated via natural killer cell activation. PLoS ONE, 13(1), e0190860.

+ Open protocol

+ Expand

Assaying CD8+ T-cell Degranulation via CD107a

Check if the same lab product or an alternative is used in the 5 most similar protocols

CD107a, also known as lysosomal-associated membrane protein 1 (LAMP1), is a surrogate for antigen-specific CD8⁺ T-cell degranulation, a hallmark of CD8⁺ T-cell cytolytic activity (25 –27 (link)). CD107a is present on the membrane of lysosomes containing perforin and granzymes in CD8⁺ T cells. When lytic granules are released, lysosomal membranes fuse with the plasma membrane resulting in cell-surface CD107a expression on CD8⁺ T cells that can be assayed using flow cytometry (25 –27 (link)). After resting overnight, PBMCs (n = 10 healthy donors, n = 10 from STAMP, and n = 12 from STRIDE) were counted and diluted to a concentration of ~2 × 10⁷ cells/mL in warm cRPMI. Approximately 2 × 10⁶ cells (100 mL of cell suspension) were placed into each well of a 96-well cell culture plate, and cells were stimulated with an equal volume of antigen [HER500, a truncated form of HER2/neu as additional negative control (20 μg/mL), PA2024 (100 μg/mL) or PAP (50 μg/mL)] at 37°C in a 5% CO₂ atmosphere for 18 to 20 hours, along with Protein Transport Inhibitor Cocktail (500×; ThermoFisher Scientific; #00–4980-03), and anti-CD107a antibody (BD Biosciences; cloneH4A3, #561343) added during the last 4 hours of culture.

Antonarakis E.S., Small E.J., Petrylak D.P., Quinn D.I., Kibel A.S., Chang N.N., Dearstyne E., Harmon M., Campogan D., Haynes H., Vu T., Sheikh N.A, & Drake C.G. (2018). Antigen-Specific CD8 Lytic Phenotype Induced by Sipuleucel-T in Hormone-Sensitive or Castration-Resistant Prostate Cancer and Association with Overall Survival. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(19), 4662-4671.

+ Open protocol

+ Expand

Multiparameter Flow Cytometry of Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

We stained the cells with the appropriate antibodies and fixed them for 1 h in 1% paraformaldehyde-containing phosphate-buffered saline (PBS) at 4 °C. We obtained the data using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed it using FlowJo v10 (BD Biosciences). We determined CD107a expression with an IMMUNOCYTO CD107a Detection Kit (MBL, Nagoya, Japan) according to the instructions.
For intracellular cytokine staining, we stimulated the cells in a 96-well round-bottom plate with T98G GBM cells at 37 °C for 4 h, then incubated them for 5 h at 37 °C with Protein Transport Inhibitor Cocktail (Thermo Fisher Scientific). Then, we fixed and permeabilized the cells with BD Cytofix/Cytoperm Kit (BD Biosciences) and incubated them for 30 min with anti-cytokine antibodies on ice. Phosphorylation was detected with PerFix-EXPOSE (Beckman Coulter, Brea, CA, USA) according to the instructions. The Supplementary Materials and Methods list the antibodies used for the flow cytometry.

Nakazawa T., Morimoto T., Maeoka R., Matsuda R., Nakamura M., Nishimura F., Ouji N., Yamada S., Nakagawa I., Park Y.S., Ito T., Nakase H, & Tsujimura T. (2023). CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma. Journal of Experimental & Clinical Cancer Research : CR, 42, 205.

+ Open protocol

+ Expand

Multiparameter Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

BD LSRII Fortessa flow cytometer was used to acquire cells. Cells were stained with the following monoclonal antibodies anti- H-2K^d (SF1–1.1.1), CD4 (RM4–5/GK1.5), CD8α (53–6.7), CD8β (H35–17.2), CD25 (PC61.5), Lag3 (C9B7W), PD-1 (J43), Tim3 (RMT3–23), Foxp3 (FJK-16s), CCR9 (CW-1.2), α4β7 (DATK-32), IFN-γ (XMG1.2), IL-17 (eBio17B7) and TNF-α (MP6-XT22). Antibodies were purchased either from Thermo Fischer Scientific (Waltham, MA) and Biolegend (San Diego, CA). Cells were stimulated with cell stimulation cocktail and protein transport inhibitor cocktail (Thermo Fischer Scientific, Waltham, MA) for 5 hours to measure intra-cellular cytokines. For intracellular staining, cells were fixed using the Foxp3/Transcription factor staining buffer set or IC fixation kit (Thermo Fischer Scientific, Waltham, MA). Active caspase-3 was measured by CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Thermo Fischer Scientific; Waltham, MA). Cells were stained with Fixable Viability Dye ef780 (Thermo Fischer Scientific, Waltham, MA) to exclude dead cells for all experiments. Data were analyzed with FlowJo (Tree Star Inc., Ashland, OR).

Thangavelu G., Lee Y.C., Loschi M., Schaechter K.M., Feser C.J., Koehn B.H., Nowak E.C., Zeiser R., Serody J.S., Murphy W.J., Munn D.H., Chambon P., Noelle R.J, & Blazar B.R. (2019). Dendritic Cell Expression of Retinal Aldehyde Dehydrogenase-2 Controls Graft-Versus-Host Disease Lethality. Journal of immunology (Baltimore, Md. : 1950), 202(9), 2795-2805.

+ Open protocol

+ Expand

Quantifying Antigen-Specific T-Cell Responses

Check if the same lab product or an alternative is used in the 5 most similar protocols

Splenocytes from vaccinated and control mice were prepared and stained with CellTrace Violet dye according to the manufacturers instructions (ThermoFisher Scientific, MA, USA). One million splenocytes were then stimulated or left untreated for 4 days in wells of a flat bottom 96 well plate in duplicate. After 4 days, the cells were restimulated with antigen for a further 24 h. At 6–8 h prior to harvesting, protein transport inhibitor cocktail was added to the cells (ThermoFisher Scientific). The cells were harvested and the duplicate wells pooled into V bottom 96 well plates to reduce cell loss during the intracellular staining. The cell samples were then stained with T-cell surface markers (CD3 FITC, CD8 PerCp-Cy5.5, CD4 APC, all ThermoFisher Scientific) followed by intracellular staining of IFN-gamma (IFN-γ PE clone XMG1.2. ThermoFisher Scientific) using the intracellular fix/perm kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The samples was analyzed with a MACSQuant16 instrument (Miltenyi Biotech).

Elder E., Bangalore Revanna C., Johansson C., Wallin R.P., Sjödahl J., Winqvist O, & Mirazimi A. (2023). Protective immunity induced by an inhaled SARS-CoV-2 subunit vaccine. Vaccine.

+ Open protocol

+ Expand

Profiling Immune Cell Activation

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs were stimulated with PMA (50 ng/mL, Sigma-Aldrich) and ionomycin (1 μg/mL, Sigma-Aldrich) in the presence of protein transport inhibitor cocktail (Thermo Fisher Scientific) for 6 hours. Cells were collected and stained with BV510-conjugated anti-CD3 (catalog 740202), APC-Cy7–conjugated anti-CD4 (catalog 557871), PerCP-Cy5.5–conjugated anti-CD8 (catalog 560662), Alexa Fluor 647–conjugated anti-CD319 (catalog 564338), and BV421-conjugated γδTCR antibody (catalog 744870) for surface markers. Cells were fixed and permeabilized before staining with Alexa Fluor 488–conjugated anti-GNLY (catalog 558254) and PE-conjugated anti-GZMA (catalog 558904). After 3 washes, cells were measured using a BD flow cytometer. For MNs, cells were measured after staining with APC-Cy7–anti-CD14 (catalog 557831), BV510–anti-CD16 (catalog 563830), BV421–anti-HLA-DR (catalog 562805), APC–anti-CCR1 (catalog 362908, BioLegend), and PE–anti-CX3CR1 (catalog 355704, BioLegend). All antibodies except anti-CCR1 and anti-CX3CR1 were purchased from BD Biosciences.

Lu C., Li S., Qing P., Zhang Q., Ji X., Tang Z., Chen C., Wu T., Hu Y., Zhao Y., Zhang X., He Q., Fox D.A., Tan C., Luo Y, & Liu Y. (2023). Single-cell transcriptome analysis and protein profiling reveal broad immune system activation in IgG4-related disease. JCI Insight, 8(17), e167602.

+ Open protocol

+ Expand

Multiparameter Flow Cytometry of Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Restimulation Assay for Lung Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For restimulation experiments, P10 lungs from Vav1^iCre/+;Csf2^fl and littermate controls were collected and processed as above. In a 96-well plate, lung cells were incubated for 4 h at 37°C and 5% CO₂ in 200 μl of either complete RPMI media (RPMI-1640, 10% FBS, 1 mM Hepes, 100 U/ml/100 μg/ml Pen/Strep, and 55 µM 2-mercaptoethanol) or complete RPMI media supplemented with 1× Protein Transport Inhibitor Cocktail (Thermo Fisher Scientific; 00–4980-03), 0.05 μg/ml PMA (Sigma; P1585), and 0.5 μg/ml ionomycin (Sigma; I0634). Upon completion of the incubation, cells were washed, stained, and analyzed by flow cytometry.

Gschwend J., Sherman S.P., Ridder F., Feng X., Liang H.E., Locksley R.M., Becher B, & Schneider C. (2021). Alveolar macrophages rely on GM-CSF from alveolar epithelial type 2 cells before and after birth. The Journal of Experimental Medicine, 218(10), e20210745.

+ Open protocol

+ Expand

Characterization of Circulating Follicular Helper T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Heparin-anticoagulated mouse peripheral blood 500ul and 1640 culture medium containing 10% calf serum were mixed, 2ul of stimulant and inhibitor were added respectively (eBioscience Cell Stimulation Cocktail and eBioscience Protein Transport Inhibitor Cocktail, thermofisher) and the cells were incubated for 5 hours at 37°C in 5% CO₂ environment, then stained for cell surface and intracellular follow the order, and finally detected by flow cytometry, in which IFN-γ expressing CD4⁺CXCR5⁺ cells were defined as cTFH1, IL-4 and IL-17 expressing CD4⁺CXCR5⁺ cells were defined as cTFH2 and cTFH17 respectively.

Jin X., Chen J., Wu J., Lu Y., Li B., Fu W., Wang W, & Cui D. (2022). Aberrant expansion of follicular helper T cell subsets in patients with systemic lupus erythematosus. Frontiers in Immunology, 13, 928359.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!