Golgiplug

Manufactured by BioLegend

Sourced in United States

GolgiPlug is a cell culture reagent used to inhibit protein transport from the endoplasmic reticulum to the Golgi apparatus, thereby preventing the secretion of proteins from the cell. It is commonly used in immunological studies to accumulate intracellular proteins for detection and analysis.

Automatically generated - may contain errors

Lab products found in correlation

16 protocols using golgiplug

Cytokine Profiling of Activated γδ T Cells

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

Primary γδ T cells or polarized γδ T cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of Golgi plug (BioLegend) for 4 hours or by IL-1β (10 ng/ml) and IL-23 (10 ng/ml) in the presence of Golgi plug (BioLegend) overnight at 37°C in complete RPMI 1640 medium. For some experiments, different concentrations of inhibitors were added as indicated. Small-molecule inhibitors including 2-DG, OXA, and AGI were purchased from Sigma-Aldrich. Cells were collected, surface-stained, fixed, and then intracellularly stained with fluorochrome-labeled mAbs against cytokines or transcription factors according to the manufacturer’s instructions. Cells were acquired by FACSCanto II (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).

Chen X., Cai Y., Hu X., Ding C., He L., Zhang X., Chen F, & Yan J. (2022). Differential metabolic requirement governed by transcription factor c-Maf dictates innate γδT17 effector functionality in mice and humans. Science Advances, 8(21), eabm9120.

+ Open protocol

+ Expand

Bone Marrow Dendritic Cell Generation and Activation

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

Bone marrow‐derived DCs (BMDCs) were generated as described in Lutz et al.41 Briefly, bone marrow hematopoietic cells were differentiated in GM‐CSF (20ng/uL; Shenandoah Biotechnology Inc., Warwick, PA) in complete DC medium (CDCM), comprising RPMI 1640, 10% FCS, 2 mM L‐glutamine, 1IU/mL Pen‐Strep, 1 mM beta‐mercaptoethanol, for 7 d. For 5 mM glucose media, glucose‐free RPMI is used as a base media in addition to dialyzed FCS, and glucose added to 5 mM concentration. On day 7, DCs were washed in CDCM and cultured at 2 × 10⁵ cells per 200 µL of media. For intracellular cytokine staining, cells were activated for a total of 4 h with an addition of Golgi plug (Biolegend) after the first hour of stimulation.

Thwe P.M., Fritz D.I., Snyder J.P., Smith P.R., Curtis K.D., O'Donnell A., Galasso N.A., Sepaniac L.A., Adamik B.J., Hoyt L.R., Rodriguez P.D., Hogan T.C., Schmidt A.F., Poynter M.E, & Amiel E. (2019). Syk‐dependent glycolytic reprogramming in dendritic cells regulates IL‐1β production to β‐glucan ligands in a TLR‐independent manner. Journal of Leukocyte Biology, 106(6), 1325-1335.

+ Open protocol

+ Expand

Bone Marrow-Derived Dendritic Cell Generation

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

Bone marrow derived DCs were generated as described in Lutz et al. (41 (link)). Briefly, bone marrow hematopoietic cells were differentiated in GM-CSF (20ng/uL; Peprotech) in complete DC medium (CDCM), comprised of RPMI1640, 10% FCS, 2mM L-glutamine, 1IU/mL Pen-Strep, 1mM beta-mercaptoethanol, for 7 days. For 5 mM glucose media, glucose-free RPMI is used as a base media in addition to dialyzed FCS, and glucose added to 5 mM concentration. On day 7, DCs were washed in CDCM and cultured at 2×10^5 cells per 200μL of media. For intracellular cytokine staining, cells were activated for a total of 4 hours with an addition of Golgi plug (Biolegend) after the first hour of stimulation.

+ Open protocol

+ Expand

DC Activation and T Cell Responses

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

DCs were treated with OVA peptide (SIINFEKL; 1.1 nM) for 30 minutes and then stimulated with β-glucan particles or LPS for 24 hours. DCs were then washed thoroughly and CFSE-labeled naïve CD8 T cells were added at a 1:5 ratio (8×10⁴ DCs plus 40×10⁴ T cells) and cultures were supplemented with 1 M sodium pyruvate and 50 mM β-mercaptoethanol. In some experiments, anti-IFNAR1 blocking antibodies, anti-CD86 blocking antibodies or isotype control antibodies (200 μg/ml; BioLegend) were added to DC cultures and/or DC:T cell co-cultures as described in the figure legends. Following 3 days of co-culture, T cells were harvested for flow cytometry (CFSE, CD44, CD69) and for re-stimulation to assess cytokine and granzyme B production. T cells were re-stimulated with 50 ng/ml PMA plus 500 ng/ml ionomycin for 6 hours with GolgiPlug and GolgiStop (Biolegend) for the last 4 hours in order to assess cytokine and granzyme B production by intracellular flow cytometry, and for 24 hours (without the protein export inhibitors) for cytokine assessment by ELISA.

Hassanzadeh-Kiabi N., Yáñez A., Dang I., Martins G.A., Underhill D.M, & Goodridge H.S. (2016). Autocrine type I IFN signaling in dendritic cells stimulated with fungal β-glucans or lipopolysaccharide promotes CD8 T cell activation. Journal of immunology (Baltimore, Md. : 1950), 198(1), 375-382.

+ Open protocol

+ Expand

Cytokine Secretion Analysis in Mice

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

To detect in situ cytokine secretion, mice were slowly i.v injected with 500 μg of Brefeldin A in 250 μl PBS 6 h before sacrifice and intracellular cytokine staining.
To detect cytokine secretion in T cells upon ex vivo re-stimulation, single cell suspensions from tumors and LNs were resuspended in RPMI 1640 with 10% FCS and added to αCD3 (clone 145–2C11)/αCD28 (clone 37.51) antibody-coated (overnight at 10 μg/ml antibody) tissue culture plates for 8 hours at 37°C in the presence of 1 μg/mL Golgiplug and Monensin (both from Biolegend) and cells processed for intracellular cytokine staining.

Pilato M.D., Kim E.Y., Cadilha B.L., Pruessmann J.N., Nasrallah M.N., Seruggia D., Usmani S.M., Misale S., Zappulli V., Carrizosa E., Mani V., Ligorio M., Warner R.D., Medoff B.D., Marangoni F., Villani A.C, & Mempel T.R. (2019). Targeting the CBM complex causes Treg cells to prime tumors for immune checkpoint therapy. Nature, 570(7759), 112-116.

+ Open protocol

+ Expand

Multiparametric Flow Cytometry of Immune Cells

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

After Zombie™ dye and Fc‐block (both BioLegend, San Diego, CA, USA) pre‐incubation, PBMC were stained with anti‐human CD14‐FITC, CD19‐PerCP‐Cy5.5, CD40‐PE‐Dazzle, CD69‐FITC, CD86‐BV421, CD95‐PE (all BD Bioscience, NJ, USA), CD4‐PE‐Cy7, CD8‐PE, CD14‐PE‐CF594, CD19‐FITC, CD20‐APC‐Cy7, CD24‐PerCp‐Cy5.5, CD25‐BV605, CD27‐PacificBlue, CD38‐FITC, CD80‐PE‐Cy7 and major histocompatibility complex (MHC)‐II‐APC (all BioLegend, CA, USA). Cytokines were evaluated after adding 1‐µL Golgi‐Plug (BioLegend) to CpG‐stimulated cells for 4 h; 500 ng/mL ionomycin and 20 ng/mL phorbol 12‐myristate 13‐acetate (PMA; Sigma Aldrich, MO) were added after 2 h. Cells were stained with anti‐human interleukin‐ (IL‐)6‐FITC, tumor necrosis factor (TNF)‐A700 and IL‐10‐PE‐CF594 (all BioLegend).

Traub J., Traffehn S., Ochs J., Häusser‐Kinzel S., Stephan S., Scannevin R., Brück W., Metz I, & Weber M.S. (2019). Dimethyl fumarate impairs differentiated B cells and fosters central nervous system integrity in treatment of multiple sclerosis. Brain Pathology, 29(5), 640-657.

+ Open protocol

+ Expand

Multiparametric flow cytometry analysis of T cell activation

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

T cells from various experiment groups: T₀, T_NPP12 and T_PP12 were cultured in complete RPMI medium, or with T2 cell (pulse with synthetic peptide pool for 2 h on rotator), or with peptide pool for 4 h, followed by adding GolgiPlug (Biolegend, San Diego, CA). To exclude dead cells, samples were stained with Live/Dead dye (Biolegend, San Diego, CA), followed by surface staining (fluorochrome-conjugated antibodies: anti-CD3, anti-CD4, and anti-CD8, Biolegend, San Diego, CA) for 30 min on ice. Cells were then fixed with Fix & Perm Cell Fixation & Permeabilization Kit (ThermoFisher) according to the instruction, stained with fluorochrome-conjugated anti-IFN-γ and anti-IL-2 (Biolegend, San Diego, CA) for 30 min on ice, washed and resuspended in staining buffer with 1% PFA, followed by flow cytometry analysis by BD LSR Fortessa (Beckman Dickinson, Franklin Lakes, NJ.). Flow cytometry data was analyzed by FlowJo (Three Star, Ashland, OR).

Miyaguchi K., Wang H., Black K.L., Shiao S.L., Wang R, & Yu J.S. (2023). Activated T cell therapy targeting glioblastoma cancer stem cells. Scientific Reports, 13, 196.

+ Open protocol

+ Expand

Stimulation and Flow Cytometry Analysis

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

To prepare for flow cytometry analysis, single-cell suspensions from the spleens and lungs were stimulated with phorbol myristate acetate (PMA) at 10 ng/mL and ionomycin at 1500 ng/mL. The cells were then placed in an incubator at 37 °C and Brefeldin A (GolgiPlug, Biolegend Cat#420601) was added at a 1000× dilution after one hour. Following an additional four hours, the cells were washed with phosphate-buffered saline and stained for analysis using flow cytometry.

Chan L., Mehrani Y., Minott J.A., Bridle B.W, & Karimi K. (2023). Dendritic Cell Vaccines Impact the Type 2 Innate Lymphoid Cell Population and Their Cytokine Generation in Mice. Vaccines, 11(10), 1559.

+ Open protocol

+ Expand

Polarized γδ T Cell Cytokine Profiling

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

Polarized CD27⁻ γδ T cells were cultured in RPMI 1640 medium with 20% fetal bovine serum (FBS), and fluorescein isothiocyanate (FITC)–conjugated control siRNA (Santa Cruz Biotechnology, sc-36869) and IDH2 siRNA (Thermo Fisher Scientific, AM16708) were transfected with Lipofectamine 2000 (11668-027, Invitrogen). For the cytokine stimulation, cells were cultured for additional 24 hours with IL-1β (10 ng/ml) and IL-23 (10 ng/ml). The Golgi plug was added in the final 4 hours. For the PMA stimulation, cells were cultured for additional 24 hours and stimulated with PMA and ionomycin in the presence of Golgiplug (BioLegend) for 4 hours. Intracellular cytokine staining of IL-17 was performed. For lentivirus infection, polarized CD27⁺ γδ T cells were cultured in complete RPMI 1640 medium with polybrene (8 μg/ml). Cells were infected with GFP-tagged blank lentivirus or IDH2 lentivirus (Applied Biological Materials Inc.) by centrifugation at the speed of 2300 rpm for 90 min at 30°C. Cells were then cultured in RPMI 1640 medium containing 20% FBS, IL-2 (10 ng/ml), and IL-7 (10 ng/ml). On the second day, GFP⁺ γδ T cells were sorted and cultured for an additional 48 hours, and intracellular IFN-γ was examined.

+ Open protocol

+ Expand

Combination Therapy of Oncolytic Virus and CAR-NK Cells in Glioma Mouse Model

Cited 2 times

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

6–8-week-old male C57BL/6 mice were implanted with the murine CT2A cell line expressing human EGFR (CT2A-hEGFR). Five days after the CT2A-hEGFR cells were implanted, mice were treated with 2 × 10⁵ pfu OV-IL15C alone, 1 × 10⁶ frozen and unsorted EGFR-CAR NK cells alone, the combination of the two agents, or saline alone. Three days after the treatment, mice were sacrificed to harvest brain tissues to isolate mononuclear cells, as previously described (22 (link),23 (link)). The mononuclear cells were subjected to assess NK and T cell infiltration or cultured with PMA (BioLegend) and 1 mg/ml of GolgiPlug^™ for 4 hours before assessing the activation capacity of these cytolytic cells by flow cytometry.

Ma R., Lu T., Li Z., Teng K.Y., Mansour A.G., Yu M., Tian L., Xu B., Ma S., Zhang J., Barr T., Peng Y., Caligiuri M.A, & Yu J. (2021). An oncolytic virus expressing IL-15/IL-15Rα combined with off-the-shelf EGFR-CAR NK cells targets glioblastoma. Cancer research, 81(13), 3635-3648.

+ 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!