For assaying IFNγ production, 4 × 105 PBMCs were stimulated for 5 h in the presence of 2 ng/mL PMA, 4 μg/mL Ca2+ ionomycin (Sigma-Aldrich), and Brefeldin A (BD GolgiPlug; BD Biosciences, San Jose, CA, USA) adapted from described stimulation conditions [32 (link)] or overnight with 40 μg/mL T. cruzi lysate at 37 °C. Brefeldin A was added 5 h prior to end of incubation, and the cells were stained with anti-CD8-Pacific Blue and anti-CD4-FITC (AbD Serotec, Raleigh, NC, USA) followed by intracellular staining with anti-bovine IFN-γ AF647 (AbD Serotec) according to the BD Cytofix/Cytoperm kit (BD Biosciences). Samples were fixed in 2% formaldehyde prior to analysis by flow cytometry.
Brefeldin a
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Brefeldin A is a chemical compound used in cell biology research. It is a fungal metabolite that disrupts the secretory pathway in eukaryotic cells by inhibiting the exchange of materials between the endoplasmic reticulum and the Golgi apparatus.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Ca2 ionomycin, by Merck Group (2 mentions)
Anti cd8 pacific blue, by Bio-Rad (2 mentions)
Anti cd4 fitc, by Bio-Rad (2 mentions)
Anti cd3, by Bio-Rad (1 mentions)
Leucoperm a, by Bio-Rad (1 mentions)
Leucoperm b, by Bio-Rad (1 mentions)
3 protocols using brefeldin a
1
Quantifying IFN-γ Production in T Cells
2
Canine Vaccine IFNγ Response Assay
3
Intracellular Cytokine Profiling in Spleen
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The measurement of intracellular cytokines in the spleen was done as per manufacturer's (BD, USA) protocol as mentioned earlier [21 (link)]. Briefly, splenocytes (4 × 106/mL) were incubated with brefeldin A (10 μg/mL) (Serotec, UK) in dark for 6 h at 37°C and reincubated with mouse Seroblock FcR at RT for another 10 min. Cells were washed and incubated with FITC-rat anti-mouse CD4+ antibody. Leucoperm A and Leucoperm B (Serotec, UK) were added at RT for 15 min and cells were dispensed in four tubes each containing 1 × 106 cells/100 μL. PE-rat anti-mouse monoclonal antibodies to cytokines interleukin- (IL-) 2, IL-4, IL-10, and IFN-γ were added to separate tubes and cells were finally suspended in 500 μL of 0.5% paraformaldehyde for flow cytometer readings.
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