In order to determine whether the mRNA we were detecting correlated with the cells producing the protein, PBMCs were stimulated overnight with either HIV-Gag peptide pools, SEB or PMA/Ionomycin. Cells were then labeled with IFNγ catch reagent using the Miltenyi IFNγ capture assay kit and protocol. Cells were allowed to secrete cytokines for 45 minutes before commencing staining. Cells were stained with viability dye and surface antibodies [CD3-BV711 (5 µl per 100 µl of sample, clone: OKT3), CD4-BV421 (5 µl per 100 µl of sample, clone: OKT4), CD8-V500 (5ul per 100 µl of sample, clone: SK1) and CD69-BV650 (5 µl per 100 µl of sample, clone: FN50)], and the flow-FISH was followed. After completing washes outlined at the end of the protocol, cells were labeled with anti-IFNγ PE detection reagent following the Miltenyi protocol. The PE detection reagent was added after the flow-FISH protocol was completed because, if added before the fixation and permeabilization steps, the methanol would have degraded the protein dye and no IFNγ protein signal would have been observed. Cells were then analyzed on the LSR Fortessa.
Anti ifnγ pe detection reagent
Manufactured by Miltenyi Biotec
The Anti-IFNγ PE detection reagent is a laboratory tool used to detect and quantify the presence of interferon-gamma (IFNγ) in biological samples. It consists of a fluorescently-labeled antibody that specifically binds to IFNγ, allowing for its identification and measurement.
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Lab products found in correlation
2 protocols using anti ifnγ pe detection reagent
1
Correlating mRNA and Protein Production
2
Correlating mRNA and Protein Production
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In order to determine whether the mRNA we were detecting correlated with the cells producing the protein, PBMCs were stimulated overnight with either HIV-Gag peptide pools, SEB or PMA/Ionomycin. Cells were then labeled with IFNγ catch reagent using the Miltenyi IFNγ capture assay kit and protocol. Cells were allowed to secrete cytokines for 45 minutes before commencing staining. Cells were stained with viability dye and surface antibodies [CD3-BV711 (5 µl per 100 µl of sample, clone: OKT3), CD4-BV421 (5 µl per 100 µl of sample, clone: OKT4), CD8-V500 (5ul per 100 µl of sample, clone: SK1) and CD69-BV650 (5 µl per 100 µl of sample, clone: FN50)], and the flow-FISH was followed. After completing washes outlined at the end of the protocol, cells were labeled with anti-IFNγ PE detection reagent following the Miltenyi protocol. The PE detection reagent was added after the flow-FISH protocol was completed because, if added before the fixation and permeabilization steps, the methanol would have degraded the protein dye and no IFNγ protein signal would have been observed. Cells were then analyzed on the LSR Fortessa.
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