Golgiplug protein transport inhibitor

Manufactured by BD

Sourced in United States

GolgiPlug is a protein transport inhibitor that blocks the secretory pathway by preventing the release of proteins from the Golgi apparatus. It is a commonly used tool in cell biology research to study intracellular protein trafficking and secretion.

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Lab products found in correlation

Cytofix cytoperm, by BD (7 mentions) Ionomycin, by Merck Group (6 mentions) Perm wash buffer, by BD (6 mentions) Phorbol myristate acetate (pma), by Merck Group (5 mentions) Cytofix cytoperm kit, by BD (5 mentions) Cytofix cytoperm solution, by BD (4 mentions) Fixation permeabilization solution kit, by BD (3 mentions) Golgistop, by BD (3 mentions) Efluor 670, by Thermo Fisher Scientific (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) Anti mouse tnf α, by BD (2 mentions) Magnetic beads, by BD (2 mentions) Anti mouse ifn γ, by BD (2 mentions) Anti mouse cd8, by BioLegend (2 mentions) Trustain fcx, by BioLegend (2 mentions) Lsr 2, by BD (2 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions) Diva software, by BD (2 mentions) Apc conjugated anti ifn γ, by BD (2 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

GolgiPlug (1864 citations) Protein transport inhibitor (20 citations) BD GolgiPlug protein transport inhibitor containing brefeldin A (7 citations) BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit with BD GolgiPlug Protein transport inhibitor containing Brefeldin A (2 citations)

49 protocols using golgiplug protein transport inhibitor

Lymph Node Analysis of Lm + MAX Challenged Mice

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Mice were vaccinated followed by a boost two weeks later. One week following boost, mice were either unchallenged or challenged with Lm + MAX. One week later (4 weeks after initial vaccination) the popliteal and/or paraaortic/lumbar lymph nodes (LN) were harvested (n = 5–8) and mechanically disrupted to prepare separate single-cell suspensions in complete RPMI medium. 1 × 10⁶ cells were added to each well of a 48-well plate in a volume of 500 µL of complete RPMI medium. 1 µL of both Cell Stimulation Cocktail (500x, eBioscience, San Diego, CA) and GolgiPlug™ Protein Transport Inhibitor (BD Biosciences Pharmingen, San Diego, CA) were added to each well, and cells were then incubated for 5 h at 37 °C. Cells were processed according to manufacturer’s protocol.

Wheat W.H., Arthun E.N., Spencer J.S., Regan D.P., Titus R.G, & Dow S.W. (2017). Immunization against full-length protein and peptides from the Lutzomyia longipalpis sand fly salivary component maxadilan protects against Leishmania major infection in a murine model. Vaccine, 35(48Part B), 6611-6619.

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Activation of NK cells by cytokine stimulation

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24-well plates were coated overnight at 4°C in PBS with 20 μg/ml purified anti-NK1.1 (PK136, eBiosciences) and anti-NKp46 (polyclonal, R&D systems). A single cell splenocyte suspension was subsequently prepared and NK cells were enriched using a negative selection kit (MACS Miltenyi Biotec). NK cells (4×10 to 5×10 cells) were stimulated for 6hours at 37 C in RPMI+10% FCS. CD107a (BD Pharmingen) was added (1:300 dilution) to the culture during stimulation. After one hour of stimulation, GolgiPlug™ Protein Transport Inhibitor diluted 1:1000 (BD Pharmingen) was dispensed into the culture. 1 μg/ml of phorbol-12-myristate-13-acetate (PMA; SIGMA) plus 0.25 μg/ml of ionomycin (SIGMA) was used for positive control and PBS alone for negative control. For cytokine stimulation, IL-18 (1 ng/ml) plus IL-12 (1 ng/ml) was used. After stimulation, staining was performed as described above. For intracellular staining, cells were fixed and then permeabilized using Cytofix/Cytoperm kit (BD Pharmingen), followed by staining with anti-IFN-γ (XMG1.2).

Ganesan S., Thanh T.L., Kadri N., Chambers B.J., Meinke S., Brodin P., Vivier E., Wetzel D.M., Koleske A.J, & Höglund P. (2017). The Abl-1 kinase is dispensable for NK cell inhibitory signaling and is not involved in murine NK cell education. Scandinavian journal of immunology, 86(3), 135-142.

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Murine T-Cell Activation and Expansion

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Golgi Plug Protein Transport Inhibitor, Cytofix/Cytoperm Solution, PermWash buffer and 70 µm cell strainer were purchased from BD Pharmingen (Franklin Lakes, USA). Click.iT EdU flow cytometry assay kit and sytox AADvanced from Life Technologies Corporation (Carlsbad, USA). Murine MicroBeads CD4 (L3T4), CD8α (Ly2) and the CD4/CD8 T-cell activation/Expansion Kit mouse were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). LPS (L2880) were purchased from Sigma-Aldrich (St.Louis, USA). Vetbond tissue adhesive from 3 M (St. Paul, USA). EDTA-capillary and Li-Heparin-capillary tubes were acquired from Sarstedt (Nümbrecht, Germany). RPMI 1640 medium from Biochrome AG (Berlin, Germany). FCS from Biowest LLC (Kansas City, USA). 2-Mercapto-ethanol from Roth (Karlsruhe, Germany). VitaLyse Lysing Buffer from BioE (St. Paul, USA). Streptavidin from Dianova (Hamburg, Germany). 24-well plates from Corning Incorporated (Corning, USA). CFSE from Enzo Lifescience Inc. (Lörrach, Germany). Lauryl-maltoside (n-Dodecyl-ß-D-maltoside, ULTROL Grade) from Calbiochem (Bad Soden, Germany). CpG (TCCATGACGTTCCTGACGTT) was acquired from Sigma-Aldrich (St.Louis, USA). GP33–41 (KAVYNFATM) and GP61–80 (GLKGPDIYKGVYQFKSVEFD) peptides were synthesized by Bio-Synthesis (Louisville, USA).

Markwart R., Condotta S.A., Requardt R.P., Borken F., Schubert K., Weigel C., Bauer M., Griffith T.S., Förster M., Brunkhorst F.M., Badovinac V.P, & Rubio I. (2014). Immunosuppression after Sepsis: Systemic Inflammation and Sepsis Induce a Loss of Naïve T-Cells but No Enduring Cell-Autonomous Defects in T-Cell Function. PLoS ONE, 9(12), e115094.

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Cytokine Secretion Assays for Splenocytes

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Splenocytes were isolated and counted as above. For PMA/ionomycin stimulation, 10⁶ splenocytes were cultured in 1 mL of complete RPMI in a 24-well plate with 50 ng/mL phorbol myristate acetate (PMA, Sigma, St. Louis, MO), 0.75 μg/mL ionomycin calcium salt (Sigma, St. Louis, MO) and 1 μL GolgiPlug protein transport inhibitor (BD Biosciences, San Jose, CA) for four hours at 37°C, 5% CO₂. For the PD-1 blockade assay, 10 μg/mL anti-PD-1 blocking antibody (Biolegend, San Diego, CA) or 10 μg/mL rat IgG2a, κ isotype control (Biolegend, San Diego, CA) was added to the complete RPMI along with PMA and ionomycin. For the T-cell receptor (TCR) activation assay, 96-well plates were coated with 10 μg/mL anti-mouse CD3e (eBioscience, San Diego, CA. clone 145-2C11) overnight at 4°C. The next day splenocytes (10⁶/well) were added in 200 μL T-cell medium (RPMI, 10% FBS, 55 μM 2-mercaptoethanol, 2 mM L-glutamine, 10 μg/ml gentamicin and 10 μM HEPES) along with 2 μg/mL of anti-mouse CD28 (eBioscience, San Diego, CA. clone 37.51) and 50 U/mL of recombinant human IL-2 (Peprotech, Rocky Hill, NJ) and cultured for three days at 37°C in 5% CO₂. Brefeldin A (eBioscience, San Diego, CA) was added six hours before intracellular cytokine cell staining.

Zha J., Smith A., Andreansky S., Bracchi-Ricard V, & Bethea J.R. (2014). Chronic thoracic spinal cord injury impairs CD8+ T-cell function by up-regulating programmed cell death-1 expression. Journal of Neuroinflammation, 11, 65.

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CD8+ T cell Adoptive Transfer and Analysis

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Total CD8⁺ T cells were purified from spleens and lymph nodes of 6–8wk old Klrk1^−/− or wild-type NOD mice by negative selection using magnetic beads (BD Biosciences) according to the manufacturer’s protocol. Isolated Klrk1^−/− and wild-type CD8⁺ T cells were then labeled with eFluor 670 (eBioscience) and CFSE (Thermo Fisher Scientific), respectively. The labeled cells were then mixed 1:1 and adoptively transferred via retro orbital injection into 6–8 wk old wild-type NOD recipient mice (Supplemental Fig. 2A). Total splenocytes were harvested after 7 d, incubated for 4–6 h in complete media with GolgiPlug protein transport inhibitor (BD Biosciences), followed by fixation and permeabilization using BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s directions. Cells were then stained overnight at 4°C with anti-mouse TNF-α (BD Pharmingen), anti-mouse IFN-γ (BD Pharmingen), and anti-mouse CD8 (BioLegend), and analyzed by flow cytometry. Transferred CD8⁺ T cells were analyzed by gating on live lymphocytes based on forward and side scatter, CD8⁺ cells, then either eFluor 670⁺ or CFSE⁺ cells.

Trembath A.P., Sharma N., Raju S., Polić B, & Markiewicz M.A. (2017). A Protective Role for NKG2D–H60a Interaction via Homotypic T Cell Contact in Nonobese Diabetic Autoimmune Diabetes Pathogenesis. ImmunoHorizons, 1(9), 198-212.

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Cytokine Production in Activated T Cells

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Isolated LP cells (1x10⁶) were stimulated with PMA (10ng/ml) and ionomycin (50ng/ml) in cell culture medium for 2 h. Following stimulation, GolgiPlug Protein Transport Inhibitor (1 μl/ml, BD) was added to the culture medium for another 3 h. The cells were harvested, washed with staining buffer (PBS with 5% FCS), and incubated with Fixable Viability Dye (1μl/ml, eBioscience) for 30 min for dead cell staining. Anti-CD16/32 antibodies were added to cells to block Fc Receptor. The cells were stained with APC-eFluor780 anti-mouse CD3 antibody for 30 min and washed with staining buffer two times. Intracellular staining (IL-17 and IL-22) was performed using BD Cytofix/Cytoperm Fixation/Permeabilization Kit according to the manufacture’s instruction. Flow cytometry data were collected using the FACS Canto II (BD Biosciences), and analyzed by FlowJo software (Tree Star Inc.).

Li X., Luck M.E., Hammer A.M., Cannon A.R, & Choudhry M.A. (2020). 6-Formylindolo (3, 2-b) Carbazole (FICZ)–mediated protection of gut barrier is dependent on T cells in a mouse model of alcohol combined with burn injury. Biochimica et biophysica acta. Molecular basis of disease, 1866(11), 165901.

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Multiparameter Flow Cytometry of Tumor Cells

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Renal tumors were homogenized and processed as described to yield single-cell suspensions [4 (link),29 (link),32 (link)]. Tumor-draining lymph nodes (TDLNs) from tumor-bearing mice were harvested and single-cell suspensions were generated by disruption with glass slides. Cells were stained with Zombie Aqua Fixable Viability Dye (Biolegend; San Diego, CA, USA) followed by TruStain FcX (Biolegend) to block Fc receptors. Cells were then extracellularly stained with saturating concentrations of conjugated antibodies. For IFNγ intracellular staining, cells were ex vivo stimulated with purified αCD3 and αCD28 (BioLegend) for four hours. GolgiPlug protein transport inhibitor (BD Biosciences; San Jose, CA, USA) was added for the final two hours. Cells were harvested and extracellularly and intracellularly stained using BD Biosciences Fixation/Permeabilization Solution Kit. Annexin V staining was done in accordance with the BD Annexin V apoptosis detection kit protocol. Results were obtained from multi-parameter flow cytometry using an Attune NxT Flow Cytometer (ThermoFisher Scientific; Waltham, MA, USA) or BD LSR II (BD Biosciences) and analyzed with FlowJo software (BD Biosciences). Doublets were excluded by FSC-A/FSC-W gating, and dead cells were excluded via Zombie Dye. Positive events were objectively determined using fluorescence minus one (FMO) controls.

Turbitt W.J., Boi S.K., Gibson J.T., Orlandella R.M, & Norian L.A. (2021). Diet-Induced Obesity Impairs Outcomes and Induces Multi-Factorial Deficiencies in Effector T Cell Responses Following Anti-CTLA-4 Combinatorial Immunotherapy in Renal Tumor-Bearing Mice. Cancers, 13(10), 2295.

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Differentiation of Naïve CD4+ T Cells

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WT and Stat3^WT/GOF naïve CD4⁺ T cells were purified using the “Naïve CD4+ T Cell Isolation Kit” (Miltenyi Biotec, Cat. 130-104-453), cultured in supplemented RPMI (Th0, Th1, and Treg) or supplemented IMDM (Th17) media, and stimulated with plate-bound anti-CD3 (2.5ug/mL; BioXCell, 145-2C11) and soluble anti-CD28 (1ug/mL; SouthernBiotech, PV-1). T cell differentiation conditions: Th1, anti-IL-4 (10ug/mL; BioXCell, 11B11), murine IL-2 (50ng/mL; PeproTech, Cat. 212–12), and murine IL-12 (10ng/mL; R&D Sysemts, Cat. 419-ML-010); Treg, anti-IL-4, anti-IFNγ (10ug/mL; BioXCell, R4-6A2), murine IL-2, and human TGF-β (2.5ng/mL; PeproTech, Cat. 100–21); classical Th17, anti-IL-4, anti-IFNγ, human TGF-β, and murine IL-6 (30ng/mL; PeproTech, Cat. 216–16); pathogenic Th17, anti-IL-4, anti-IFNγ, human TGF-β, murine IL-1β (20ng/mL; Miltenyi Biotec, Cat. 130-094-053), and murine IL-23 (20ng/mL; R&D Systems, Cat. 1887-ML-010); Th0, anti-IL-4, anti-IFNγ, and IL-2. On day 5, cells were stimulated with phorbol-12-myristate-13-acetate (PMA; 50ng/mL; Sigma-Aldrich, Cat. 5.00582), ionomycin (1ug/mL; MilliporeSigma, Cat. 407950), and GolgiPlug Protein Transport Inhibitor (1:1000; BD Biosciences, Cat. 555029) and then stained for flow cytometry.

Woods J., Pemberton S.E., Largent A.D., Chiang K., Liggitt D., Oukka M., Rawlings D.J, & Jackson S.W. (2022). Systemic autoimmunity in murine STAT3 gain-of-function syndrome is characterized by effector T cell expansion, in the absence of overt regulatory T cell dysfunction. Journal of immunology (Baltimore, Md. : 1950).

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Expansion and Characterization of HIV-Specific CD8+ T Cells

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B-EBV lines were generated for each donor by culturing PBMCs in RPMI, 20% fetal bovine serum (FBS), 20nM FK506 (AG Scientific), and Ebstein-barr virus (EBV)-containing supernatant from the virus-producing B95.8 marmoset cell line (ATCC) at an MOI of 100. B-EBV cell lines were then loaded with 0.5–5 mg/mL HIV Clade AE peptide pools overnight. Peptide pools were made with 20 15-mer peptides per pool of HIV PTE and HIV Consensus A peptides (gag, pol, nef, env) obtained through the AIDS Reagent Program, Division of AIDS, NIAID. Expanded primary CD8+ T cells were added to the loaded B-EBV (2:1), co-stimulated with 1 μg/μL αCD28/CD49d (BD Biosciences) and incubated for 12 hours with GolgiPlug protein transport inhibitor (BD Biosciences). After incubation, cells were stained with αCD8-FITC, αCD3-PacificBlue (BD Biosciences), and αCD20-PECy7 (BioLegend) prior to fixation/permeabilization and intracellular staining with αIFNγ-APC (BD Biosciences). Live/dead stain with Vivid-amcyan was used to exclude dead cells from the analysis. The T-cell receptor Vbeta repertoire was analyzed by flow cytometry using the IOTest^® Beta mark (Beckman Coulter) in conjunction with αCD8-Pacific Blue (BD Biosciences). Stained CD8+ T cells were run on an LSRII flow cytometer using DiVA software (BD Biosciences) and analyzed with FlowJo (Treestar).

Kessing C.F., Spudich S., Valcour V., Cartwright P., Chalermchai T., Fletcher J.L., Nichols C., Josey B.J., Slike B., Krebs S.J., Sailsuta N., Lerdlum S., Jagodzinski L., Tipsuk S., Suttichom D., Rattanamanee S., Zetterberg H., Hellmuth J., Phanuphak N., Robb M.L., Michael N.L., Ananworanich J, & Trautmann L. (2017). High Number of Activated CD8+ T Cells Targeting HIV Antigens are Present in Cerebrospinal Fluid in Acute HIV Infection. Journal of acquired immune deficiency syndromes (1999), 75(1), 108-117.

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Flow Cytometry for Immune Profiling

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To block Fc receptors, human samples were incubated in 5% normal rat serum and murine samples were incubated in TruStain FcX (BioLegend) and normal rat serum then stained using antibodies listed in online supplemental table 4. Intracellular staining of murine Foxp3 was performed according to manufacturer instructions using a Foxp3/Transcription factor staining set (ThermoFisher). For murine IFNγ, TNF⍺ and perforin intracellular staining, bulk tumor cells were ex vivo stimulated for 4 hours with plate-bound, purified anti-CD3 and anti-CD28 (BioLegend). GolgiPlug protein transport inhibitor (BD Biosciences) was added for the final 2 hours. Cells were harvested and stained using BD Biosciences Fixation/Permeabilization Solution Kit. Results were obtained using an Attune NxT (ThermoFisher) or BD LSR II (BD Biosciences) flow cytometer and analyzed with FlowJo Software. Dead cells were excluded via Zombie Aqua or Green Fixable Viability Dye (BioLegend). Fluorescence minus one controls were used to objectively determine gate boundaries for positive events.

Boi S.K., Orlandella R.M., Gibson J.T., Turbitt W.J., Wald G., Thomas L., Buchta Rosean C., Norris K.E., Bing M., Bertrand L., Gross B.P., Makkouk A., Starenki D., Farag K.I., Sorge R.E., Brown J.A., Gordetsky J., Yasin H., Garje R., Nandagopal L., Weiner G.J., Lubaroff D.M., Arend R.C., Li P., Zakharia Y., Yang E., Salem A.K., Nepple K., Marquez-Lago T.T, & Norian L.A. (2020). Obesity diminishes response to PD-1-based immunotherapies in renal cancer. Journal for Immunotherapy of Cancer, 8(2), e000725.

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