Monensin and brefeldin a

Manufactured by Thermo Fisher Scientific

Sourced in United States

Monensin and brefeldin A are laboratory reagents used in cell biology research. Monensin is a carboxylic ionophore that disrupts intracellular protein transport. Brefeldin A is a fungal metabolite that inhibits the function of the Golgi apparatus, affecting intracellular protein trafficking. These reagents are widely used to study cellular processes involving membrane trafficking and secretion.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

Brefeldin A (734 citations) Monensin (334 citations) Brefeldin A/monensin (3 citations) Golgi stop (brefeldin A and monensin) (3 citations)

8 protocols using monensin and brefeldin a

Cytokine Production Profiling by Flow Cytometry

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

Single cell suspensions prepared in complete medium (RPMI + 10% FBS + 1 mM Sodium pyruvate) were incubated with 50 nM phorbol 12, 13-dibutyrate (PDBu) and 0.5 μg/ml ionomycin (Sigma) for 4 h before further processing including measurement of cytokine production by flow cytometry or Flow-FISH.
For intracellular cytokine detection monensin and brefeldin A (eBioscience) were added during the last 2 h of the stimulation. Staining was done as described in the previous section and the following antibodies were used: anti-human TNF (clone Mab11, BD), anti-human IL-10 (clone JES3–19F1, Biolegend) and anti-bat IFN-γ (clone 2G645 (link), a kind gift from CSIRO) + F(ab’)2 anti-mouse IgG PE-conjugated (eBioscience) (Table S2).

Martínez Gómez J.M., Periasamy P., Dutertre C.A., Irving A.T., Ng J.H., Crameri G., Baker M.L., Ginhoux F., Wang L.F, & Alonso S. (2016). Phenotypic and functional characterization of the major lymphocyte populations in the fruit-eating bat Pteropus alecto. Scientific Reports, 6, 37796.

+ Open protocol

+ Expand

Flow Cytometry Analysis of Murine Splenocytes

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

Mice splenocytes were incubated with Fc‐receptor blocking antibodies (2.4G2; BD Biosciences) for 10 min on ice to block non‐specific staining. For surface staining, fluorophore‐conjugated anti‐mouse antibodies were incubated in PBS containing 2% heat‐inactivated foetal bovine serum (Gibco, Carlsbad, California, USA) for 30 min. For staining of intracellular cytokines, whole splenocytes were stimulated with PMA and ionomycin (500 ng mL⁻¹; eBioscience, San Diego, California, USA) in the presence of monensin and brefeldin A (1:1000; eBioscience) for 4 h at 37°C. Stimulated cells were washed twice after surface staining and permeabilised using Cytofix/Cytoperm (BD Bioscience) for 30 min at 4°C. Antibodies specific for intracellular cytokines were incubated with cells for 30 min at 4°C. Isotype controls were used as negative controls. To detect endogenous LCMV‐specific CD8⁺ T cells, APC‐conjugated H‐2D^b‐gp33‐tetramer (MBL, TS‐M512‐P) was used. The stained cells were analysed using a BD LSRFortessa™ X‐20 (BD Bioscience) instrument and FlowJo software (TreeStar, Ashland, OR, USA).

Xie M., Yunis J., Yao Y., Shi J., Yang Y., Zhou P., Liang K., Wan Y., Mehdi A., Chen Z., Wang N., Xu S., Zhou M., Yu M., Wang K., Tao Y., Zhou Y., Li X., Liu X., Yu X., Wei Y., Liu Z., Sprent J, & Yu D. (2021). High levels of soluble CD25 in COVID‐19 severity suggest a divergence between anti‐viral and pro‐inflammatory T‐cell responses. Clinical & Translational Immunology, 10(2), e1251.

+ Open protocol

+ Expand

Intracellular Cytokine Staining Assay

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

Spleen cell suspensions were cultured for 5h in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), 2 mM glutamine (Gibco), 55 μM 2-ME (Gibco), and 40 μg/ml gentamicin. Cells were stimulated with 2.5 μM TSKB20 (ANYKFTLV) peptide (Genscript Inc.) or 50 ng/mL PMA plus 1 μg/mL ionomycin (Sigma-Aldrich) in the presence of Monensin and Brefeldin A (eBioscience). Culture medium was used as negative control. Anti-CD107a was included during the culture period. After surface staining, cells were fixed and permeabilized using Intracellular Fixation & Permeabilization Buffer Set (eBioscience) following manufacturer’s instructions. Stained cells were acquired on a FACSCanto II (BD Biosciences) or a LSRFortessa X-20 (BD Biosciences) flow cytometer as before. Antibodies specifications are detailed in S1 Table.

Araujo Furlan C.L., Boccardo S., Rodriguez C., Mary V.S., Gimenez C.M., Robson S.C., Gruppi A., Montes C.L, & Acosta Rodríguez E.V. (2024). CD39 expression by regulatory T cells participates in CD8+ T cell suppression during experimental Trypanosoma cruzi infection. PLOS Pathogens, 20(4), e1012191.

+ Open protocol

+ Expand

Spleen Cell Cytokine and Cytotoxicity Assay

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

Spleen cell suspensions were cultured during 5 h with medium, 5 μg/ml TSKB20 (ANYKFTLV) peptide (Genscript Inc.), 50 nM PMA plus 0.5 μg/ml ionomycin (Sigma-Aldrich) in the presence of Monensin and Brefeldin A (eBioscience). When indicated a PE-labeled anti-CD107a mAb (eBioscience, eBio1D4B) was included during the culture period. After culture, the cells were surface stained, fixed and permeabilized with BD Cytofix/Cytoperm and Perm/Wash (BD Biosciences) according manufacturer's instruction. Cells were incubated with FITC-labeled antibodies to IFNγ (eBioscience, XMG1.2) or Perforin-1 (eBoscience, eBioOMAK-D), PerCP/PerCP-eFluor 710 labeled antibody to TNF (Biolegend, MP6-XT22) or Granzyme A (eBiosciences, GzA-3G8.5) and/or APC/AF647-labeled antibody to TNF (Biolegend, MP6-XT22) or Granzyme B (Biolegend, GB11). Stained cells were acquired on FACSCanto II (BD Biosciences).

Tosello Boari J., Araujo Furlan C.L., Fiocca Vernengo F., Rodriguez C., Ramello M.C., Amezcua Vesely M.C., Gorosito Serrán M., Nuñez N.G., Richer W., Piaggio E., Montes C.L., Gruppi A, & Acosta Rodríguez E.V. (2018). IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During Trypanosoma cruzi Infection. Frontiers in Immunology, 9, 2347.

+ Open protocol

+ Expand

Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells

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

Tumors, blood, spleen, and draining lymph nodes were harvested from mice and processed for flow cytometry analysis as previously described (27) . For surface staining, tumor-infiltrating lymphocyte or immune cell suspensions were stained with antibodies and respective isotype antibodies in the presence of anti-CD16/32 (2.4G2) to block FcR. The list of flow cytometry antibodies used is described in table S6. To stain for Ki67 and FoxP3, samples were fixed and permeabilized with the FOXP3 Fixation/Permeabilization Kit (eBioscience). To measure intracellular cytokine staining, single-cell suspensions were incubated for 5 hours in complete RPMI with monensin and brefeldin A (eBioscience). Samples were then surface stained before being fixed/permeabilized (BD CytoFix/CytoPerm Kit) and stained with anti-IFN-, anti-TNF, and anti-IL-2. All data were collected on an LSR Fortessa (Becton Dickinson) flow cytometer and analyzed with FlowJo v10 software (Tree Star Inc.). Gating strategy is shown in figs. S4 and S8.

Kaptein P., Jacoberger-Foissac C., Dimitriadis P., Voabil P., de Bruijn M., Brokamp S., Reijers I., Versluis J., Nallan G., Triscott H., McDonald E., Tay J., Long G.V., Blank C.U., Thommen D.S, & Teng M.W.L. (2022). Addition of interleukin-2 overcomes resistance to neoadjuvant CTLA4 and PD1 blockade in ex vivo patient tumors. Science translational medicine, 14(642).

+ Open protocol

+ Expand

Characterization of T cell kinetics post-viral vector

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

For the characterisation of T cell kinetics after IV viral vector, freshly isolated PBMC were stimulated with anti-CD28 and anti-CD49d at 1μg/mL (Becton Dickinson), 200μg/mL of CD107a-PeCy5 (eBioscience) together with a pool of all 56 peptides of the T9/96 strain P. falciparum TRAP antigen (final concentration 5µg/mL) for 16-20 hours (32 (link)), 5μg/ml Staphylococcal enterotoxin B (SEB) (Sigma Aldrich) or media (unstimulated). Brefeldin A and monensin (eBioscience), both at 1 μg/mL, were added after two hours. For lymphocytes used in matched PBMC and FNA characterisation, no peptide stimulation was performed.
Following surface staining, fixation and intracellular staining (see Supplementary Materials and Methods for antibody list), acquisition was performed using an LSRII or LSRFortessa X-20 SORP (BD Biosciences). At least 500,000 events were acquired per sample, with data analysed on FlowJo version 10 (BD Biosciences). Lymphocytes were gated on live, size, and singlet (FSC-A vs FSC-H and FSC-A vs FSC-W) and dead cells (Live/Dead amine reactive+), monocytes (CD14+) and B cells (CD19+) were excluded. Cells were subsequently gated on CD45+ CD3+ CD8+ T cells.

Noé A., Datoo M.S., Flaxman A., Husainy M.A., Jenkin D., Bellamy D., Makinson R.A., Morter R., Ramos Lopez F., Sheridan J., Voukantsis D., Prasad N., Hill A.V., Ewer K.J, & Spencer A.J. (2022). Deep Immune Phenotyping and Single-Cell Transcriptomics Allow Identification of Circulating TRM-Like Cells Which Correlate With Liver-Stage Immunity and Vaccine-Induced Protection From Malaria. Frontiers in Immunology, 13, 795463.

+ Open protocol

+ Expand

SARS-CoV-2 Spike-specific T-cell Immune Response

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

Spike-specific CD4 and CD8 T-cell response was measured as previously described⁴². Biobanked PBMCs were thawed in batches and re-suspended in cRPMI. Cells were incubated in the presence of SARS-CoV-2 spike peptide pools (Miltenyi PepTivator®SARS-CoV-2) at a concentration of 1 µg/mL and 5–10 × 10⁶ cells/mL. After 4 h, protein transport inhibitor of brefeldin A and monensin (eBioscience) was added to each well and samples were incubated at 37 °C for a further 12 h.
Cells were then stained for cell surface markers (CD3 VioGreen 1:100 dilution, CD4 PE-Vio 770 1:100 dilution, CD8 PerCP-Vio700 1:100 dilution, CD69 VioBlue 1:100 dilution, (Miltenyi Biotec), PD-1 BV711 1:40 dilution (BioLegend) and “fixed”, permeabilised and stained for intracellular cytokines (IFNγ Alexa Flour 488 1:40 dilution (Biolegend) and TNFα APC 1:100 dilution (Miltenyi Biotec)). Multicolour flow cytometry was performed using Attune™ NxT Flow Cytometer. The gating strategy is shown in Supplementary Fig. 2.

Kenny G., O’Reilly S., Wrigley Kelly N., Negi R., Gaillard C., Alalwan D., Saini G., Alrawahneh T., Francois N., Angeliadis M., Garcia Leon A.A., Tinago W., Feeney E.R., Cotter A.G., de Barra E., Yousif O., Horgan M., Doran P., Stemler J., Koehler P., Cox R.J., O’Shea D., Olesen O.F., Landay A., Hogan A.E., Lelievre J.D., Gautier V., Cornely O.A, & Mallon P.W. (2023). Distinct receptor binding domain IgG thresholds predict protective host immunity across SARS-CoV-2 variants and time. Nature Communications, 14, 7015.

+ Open protocol

+ Expand

PMA and Ionomycin Stimulation of PBMCs

Cited 1 time

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

PBMCs were stimulated for 3 hr with 150 ng/ml PMA + 1 μM ionomycin in RPMI plus brefeldin A and monensin (eBioscience), 2.5 μg/ml anti-CD107α, 1.25 μg/ml anti-CD107b (BD Bioscience), and 10 μM TAPI-2 (VWR International). Following stimulation, cells were resuspended in cytometry buffer (PBS + 0.05% sodium azide + 2 mM EDTA + 2% fetal calf serum) and stained with isotope-tagged antibodies before being acquired on the CyTOF. For the detailed protocol, see Newell et al. (2012 (link)) and the Supplemental Experimental Procedures.

Fergusson J.R., Smith K.E., Fleming V.M., Rajoriya N., Newell E.W., Simmons R., Marchi E., Björkander S., Kang Y.H., Swadling L., Kurioka A., Sahgal N., Lockstone H., Baban D., Freeman G.J., Sverremark-Ekström E., Davis M.M., Davenport M.P., Venturi V., Ussher J.E., Willberg C.B, & Klenerman P. (2014). CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages. Cell Reports, 9(3), 1075-1088.

+ 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!