Log In Sign up
The largest database of trusted experimental protocols

Golgiplug reagent

Manufactured by BD
Visit Supplier
Sourced in United States

GolgiPlug reagent is a cell culture product that inhibits the transport of newly synthesized proteins from the Golgi apparatus to the cell surface. It contains the active ingredient Brefeldin A, which disrupts the Golgi apparatus and blocks the secretory pathway. The GolgiPlug reagent is commonly used in cell biology research to study protein trafficking and secretion.

Automatically generated - may contain errors

Lab products found in correlation

Uv blue live dead fixable stain, by Thermo Fisher Scientific (2 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions) Facsverse, by BD (2 mentions) Lsrii flow cytometer, by Tree Star (1 mentions) Pe anti ifn γ clone xmg1.2, by BD (1 mentions) Perm wash buffer, by BD (1 mentions) Anti il 17a, by BioLegend (1 mentions) Anti ifn γ, by BioLegend (1 mentions) Canto 2, by BD (1 mentions) Canto 2 flow cytometer, by BD (1 mentions) Fitc conjugation kit, by Abcam (1 mentions) Lipopolysaccharides (lps), by BD (1 mentions) Anti cd45, by BioLegend (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions)

Close spelling variants of this product

GolgiPlug

8 protocols using golgiplug reagent

1

Cytokine Profiling of Engineered Tumor Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
PC3 or Huh-7 cells were seeded at 0.5 X 106 cells/well in a 6-well plate followed by incubation overnight. The next day, when the cells had reached around 50% confluency, the cells were transduced with LV control, LV-shFDPS or LV-shFDPS-IL2 using a multiplicity of infection (MOI) of 5 or 10. Three days after transduction, the transduced PC3 or Huh-7 cells were collected, centrifuged, and resuspended in fresh, complete DMEM medium. The PC3 or Huh-7 cells were added at 5 X 105 cells/100 µL in wells of 96-well U-bottom plates. Next, 1 X 106/100 µL Vδ2 T cells were added to the wells and cytokine secretion was arrested by adding the GolgiPlug reagent per the manufacturer’s instructions (BD Biosciences, Franklin Lakes, NJ, USA). The coculture was incubated in a 37°C cell incubator for 4 h, whereupon cells were collected and cytokine expression was measured by flow cytometry.
Liou M.L., Lahusen T., Li H., Xiao L, & Pauza C.D. (2022). Reducing farnesyl diphosphate synthase levels activates Vγ9Vδ2 T cells and improves tumor suppression in murine xenograft cancer models. Frontiers in Immunology, 13, 1012051.
+ Open protocol
+ Expand
2

Immune Cell Profiling of CNS Tissues

Check if the same lab product or an alternative is used in the 5 most similar protocols
Animals were perfused with PBS and brains and spinal cords were removed. A single cell suspension was obtained and passed through a 70 μm strainer. Mononuclear cells were obtained by Percoll gradient (37%/70%) centrifugation and collected from the interphase. For intracellular cytokine analysis, cells were washed and stimulated with 5 ng/ml of PMA, 250 ng/ml of ionomycin the presence of brefeldin A (Golgi Plug reagent, BD Bioscience) for 4 hours. Cells were labeled with the UV-Blue Live/Dead fixable stain (Invitrogen) followed by surface staining (CD45, CD11b, CD4, CD8, TCRγδ, and TCRβ). Afterwards, cells were fixed, permeabilized and stained for intracellular IL-17A, IFNγ, and GM-CSF as described above. Alternatively, unstimulated CNS cells were surface labeled for CD45, CD11b, TCRβ, CD4, CD8, then fixed and stained for FoxP3, as above.
Bearoff F., del Rio R., Case L.K., Dragon J.A., Nguyen-Vu T., Lin C.Y., Blankenhorn E.P., Teuscher C, & Krementsov D.N. (2016). Natural genetic variation profoundly regulates gene expression in immune cells and dictates susceptibility to CNS autoimmunity. Genes and immunity, 17(7), 386-395.
+ Open protocol
+ Expand
3

Quantification of Antigen-Specific T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Presence of activated antigen-specific CD8+ and CD4+ T cells after immunization was determined by flow cytometric analysis of cells after intracellular cytokine staining assay. Cell were stimulated overnight with gp140 protein and then incubated further with GolgiPlug reagent (BD Biosciences, San Jose, CA) for 6 hours prior to cellular staining. Cells were first stained for surface markers and then permeablized for staining with PE-conjugated IFN-γ antibody (BD Biosciences, San Jose, CA) in 1× Perm/Wash Buffer (BD Biosciences, San Jose, CA)(17). Samples were run on the LSRII flow cytometer and analyses were performed using FlowJo software (Tree Star Inc, Ashland, OR). The monoclonal antibodies used were PB-anti-CD3 (clone 500A2), PerCPCy5.5-anti-CD8 (clone 53-6.7) APC-anti-CD4 (clone RM4-5) and PE-anti-IFN-γ (clone XMG1.2), all purchased from BD Biosciences, San Jose, CA. For exclusion of cells with background fluorescence for PE, an aliquot of cells used for fluorescence-minus-one (FMO) from each tissue was stained with anti-CD3, anti-CD8 and anti-CD4 antibodies and used as control for establishing gates for CD4+IFN-γ+ and CD8+IFN-γ+ cell population. Percentage of antigen specific activated cells within CD4+ and CD8+ lymphocyte subsets was determined for animals receiving immunization with either gp140 protein alone or gp140 + adjuvants.
Singh S., Yang G., Byrareddy S.N., Barry M.A, & Sastry K.J. (2014). Natural Killer T Cell and TLR9 Agonists as Mucosal Adjuvants for Sublingual Vaccination with Clade C HIV-1 Envelope Protein. Vaccine, 32(51), 6934-6940.
+ Open protocol
+ Expand
4

Characterizing CNS-infiltrating Cells in EAE Mice

Check if the same lab product or an alternative is used in the 5 most similar protocols
To characterize CNS-infiltrating cells at day 30 post-EAE induction, mice were euthanized by exsanguination by transcardial perfusion with PBS under isoflurane anesthetization. Lymphocytes were isolated from the spinal cord by Dounce homogenization to generate a single-cell suspension that was filtered with a 70-μm strainer followed by Percoll gradient (37%/70%) centrifugation and collection of the interphase. For intracellular cytokine analysis, cells were stimulated with 20 ng/ml PMA, 1 μg/ml of ionomycin and 1 mg/ml brefeldin A (Golgi Plug reagent, BD Bioscience) for 4 h. Cells were stained with the UV-Blue Live/Dead fixable stain (Thermo Fisher, USA) followed by surface staining with antibodies against CD45, CD11b, CD19, TCRβ, CD4, CD8, and TCRγδ (Biolegend, USA). For intracellular cytokine staining, cells were fixed, permeabilized with 0.05% saponin, and labeled with anti-IL-17A, anti-IFNγ, and anti-GM-CSF antibodies (Biolegend, USA). Cells were analyzed using a Cytek Aurora (Cytek Biosciences, USA). Spectral unmixing was performed with appropriate single-color controls using autofluorescence correction from an unstained group control. Data were analyzed using FlowJo software, version 10.8.1 (Tree Star Inc, Ashland, OR).
Montgomery T.L., Eckstrom K., Lile K.H., Caldwell S., Heney E.R., Lahue K.G., D’Alessandro A., Wargo M.J, & Krementsov D.N. (2022). Lactobacillus reuteri tryptophan metabolism promotes host susceptibility to CNS autoimmunity. Microbiome, 10, 198.
+ Open protocol
+ Expand
5

Flow Cytometry for Immune Cell Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Flow cytometry assays were performed as previously described.28 (link) Briefly, cells were washed with phosphate-buffered saline containing 2% FBS and stained with surface antibodies. Intracellular staining was performed with specific antibodies and the FOXP3/Transcription Factor Staining Buffer set (Thermo Fisher Scientific) according to the manufacturer’s instructions. For intracellular cytokine staining, the GolgiPlug reagent (BD Biosciences) was added for the last 4 hours of culture. Samples were assessed with BD Canto II or BD FACSVerse and FlowJo software (BD Biosciences). The staining antibodies were diluted according to the manufacturer’s instructions. The antibodies used in flow cytometry are listed in online supplemental table S5.
Kawashima S., Inozume T., Kawazu M., Ueno T., Nagasaki J., Tanji E., Honobe A., Ohnuma T., Kawamura T., Umeda Y., Nakamura Y., Kawasaki T., Kiniwa Y., Yamasaki O., Fukushima S., Ikehara Y., Mano H., Suzuki Y., Nishikawa H., Matsue H, & Togashi Y. (2021). TIGIT/CD155 axis mediates resistance to immunotherapy in patients with melanoma with the inflamed tumor microenvironment. Journal for Immunotherapy of Cancer, 9(11), e003134.
+ Open protocol
+ Expand
6

Flow Cytometry Immunophenotyping Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Flow cytometry assays were performed as described (43 (link)). Briefly, cells were washed with PBS containing 2% FBS and subjected to staining with surface antibodies. Intracellular staining was performed with specific antibodies and the FOXP3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's protocol. For intracellular cytokine staining, GolgiPlug reagent (BD Biosciences) was added for the last 4 hours of culture. Samples were assessed with a BD FACSVerse instrument (BD Biosciences) and FlowJo software (BD Biosciences). The staining antibodies were diluted following the manufacturer's instructions. Supplementary Table S8 summarizes the antibodies used in the flow cytometry analyses.
Morinaga T., Inozume T., Kawazu M., Ueda Y., Sax N., Yamashita K., Kawashima S., Nagasaki J., Ueno T., Lin J., Ohara Y., Kuwata T., Yukami H., Kawazoe A., Shitara K., Honobe-Tabuchi A., Ohnuma T., Kawamura T., Umeda Y., Kawahara Y., Nakamura Y., Kiniwa Y., Morita A., Ichihara E., Kiura K., Enokida T., Tahara M., Hasegawa Y., Mano H., Suzuki Y., Nishikawa H, & Togashi Y. (2022). Mixed Response to Cancer Immunotherapy is Driven by Intratumor Heterogeneity and Differential Interlesion Immune Infiltration. Cancer Research Communications, 2(7), 739-753.
+ Open protocol
+ Expand
7

Immune Cell Profiling of CNS Tissues

Check if the same lab product or an alternative is used in the 5 most similar protocols
Animals were perfused with PBS and brains and spinal cords were removed. A single cell suspension was obtained and passed through a 70 μm strainer. Mononuclear cells were obtained by Percoll gradient (37%/70%) centrifugation and collected from the interphase. For intracellular cytokine analysis, cells were washed and stimulated with 5 ng/ml of PMA, 250 ng/ml of ionomycin the presence of brefeldin A (Golgi Plug reagent, BD Bioscience) for 4 hours. Cells were labeled with the UV-Blue Live/Dead fixable stain (Invitrogen) followed by surface staining (CD45, CD11b, CD4, CD8, TCRγδ, and TCRβ). Afterwards, cells were fixed, permeabilized and stained for intracellular IL-17A, IFNγ, and GM-CSF as described above. Alternatively, unstimulated CNS cells were surface labeled for CD45, CD11b, TCRβ, CD4, CD8, then fixed and stained for FoxP3, as above.
Bearoff F., del Rio R., Case L.K., Dragon J.A., Nguyen-Vu T., Lin C.Y., Blankenhorn E.P., Teuscher C, & Krementsov D.N. (2016). Natural genetic variation profoundly regulates gene expression in immune cells and dictates susceptibility to CNS autoimmunity. Genes and immunity, 17(7), 386-395.
+ Open protocol
+ Expand
8

Evaluating PAR1 and PAR3 Expression in Whole Blood

Check if the same lab product or an alternative is used in the 5 most similar protocols
For analysis of PAR1 and PAR3 levels in vivo, whole blood samples of healthy controls were stained with anti-CD 45 (Clone H130, APC/Cy7), anti-CD14 (Clone HCD14, PerCP), anti-CD16 (Clone 3G8, PE, all BioLegend), and the respective anti-PAR1 (clone ATAP2, fluorescein isothiocyanate [FITC]) or anti-PAR3 (clone H-103) antibodies (Santa Cruz). The anti-PAR3 antibody was labeled with a FITC conjugation kit (Abcam). Samples were processed as previously published 16 and measured on a BD Canto II flow cytometer. Note that 100,000 events were recorded and analyzed with the BD Diva 6 software. To investigate if inflammatory activation affects PAR levels on the respective cell surface, whole blood samples were incubated ex vivo with LPS (1 µg/mL, Sigma Aldrich, Missouri, United States) for 4 hours at 37°C and afterwards cells were stained and analyzed as described above. In addition, whole blood samples were incubated with 1 µL/mL GolgiPlug reagent (BD Biosciences, San Jose, California, United States) as recommended by the manufacturer for 30 minutes at 37°C prior to LPS challenge to block transport of newly produced proteins from the Golgi apparatus by brefeldin A. After the stimulation with LPS for 4 hours, blood samples were processed for flow cytometric analysis as described above. Gating was performed as published recently. 16
Thaler B., Hohensinner P.J., Baumgartner J., Haider P., Krychtiuk K.A., Schörgenhofer C., Jilma B., Hell L., Fischer M.B., Huber K., Hengstenberg C., Speidl W.S, & Wojta J. (2019). Protease-Activated Receptors 1 and 3 are Differentially Expressed on Human Monocyte Subsets and are Upregulated by Lipopolysaccharide Ex Vivo and In Vivo. Thrombosis and haemostasis, 119(9).
+ 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?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!