BioLegend
Alexa Fluor® 647 anti-mouse IFN-γ Antibody
505814
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Mouse splenocytes were activated by anti-CD3/anti-CD28 for 96 hours, in polarizing conditions towards Tc1/Th1 or Tc17/Th17, followed by restimulation with PMA/Ionomycin in presence of brefeldin-A (golgi-stop). intracellular staining for IFN-g was done to find differences in cytokine production.
Great choice if you use both more than 4 color Flow cytometer.
We are a T cell immunology lab and the aim of this experiment was to dissect differences in cytokine production between T cells polarized to Tc1, Tc17 or Treg.
Specific goal of this experiment was to identify differences in IFN-g and Il-17 production by polarized T cells. We cultured splenocytes from p53 knock-out and p53wild type mice and under different polarizing conditions. We wanted to see whether p53 deficiency caused differences in T cell polarization and functionality by way of alteration in cytokine production.
Product description and advantage: During a 7 color flow cytometry experiment, using IFN-g in A647 helps in a great way as you may still use APC for a different marker. Thus the bright staining flourochrme APC is still open along with obtaining great staining intensity with this IFN-g A647 antibody from Biolegend.(I use 1:100 dilution and get excellent staining).
Flow Cytometry
Mouse splenocytes
10 mins in staining buffer for Fc block
Fc block
30 mins in staining buffer for surface receptor staining. (CD4/CD8) followed by fixation of cells in BD fix/perm buffer for 15 mins. in ice. Then washed with BD perm/wash buffer and 30 mins in IFN-g antibody diluted in perm/wash buffer with dilution of 1:100 and kept on ice
30 mins in IFN-g antibody diluted in perm/wash buffer with dilution of 1:100 and kept on ice
Flow cytometry
Mouse splenocytes from two different strains were polarized towards Tc1/Th1 or Tc17/ Th17 and then re-stimulated for 4 hrs to find differences in cytokine production. There was a difference in amount of cytokine production after re-stimulation between the two groups as depicted by intracellular cytokine staining.
None
Good specificity, good fluorescence intensity.
Nothing much.
Good antibody to determine intracellular Interferon-gamma. It being Alexa647 gives you the advantage of using more colors if you are using a more than 4 color flow cytometer.