Whole cells were fixed with 4% paraformaldehyde in 1× PBS. Both fixed whole cells and fixed nuclei were permeabilized with 0.3% Triton X-100 in1× PBS and 2% FBS and stained with fluorescent Abs for 15 min at room temperature followed by three washes in 1× PBS and 2% FBS. To sufficiently pellet nuclei, centrifugation was performed at 1000 × g. The following Abs were used for staining: β-tubulin-AF647 (9F3, #3624; Cell Signaling Technology), CD3ε-APC (145–2C11; BD Biosciences), NFAT1-AF488 (D43B1, #14324; Cell Signaling Technology), and NF-κB (p65) (D14E12, #8242; Cell Signaling Technology). Propidium iodide was sourced from Thermo Fisher Scientific. Samples were collected on an LSR 11 flow cytometer (BD Biosciences) and analyzed with FlowJo 10.4.2 (BD Biosciences/Tree Star). After gating on singlets, OT-I nuclei were identified as CellTrace Violethi, β-tubulinlo. At least 10,000 OT-I nuclei events were recorded per sample.
Nfat1 af488 d43b1
Manufactured by Cell Signaling Technology
NFAT1-AF488 (D43B1) is a fluorescently-labeled antibody targeting the NFAT1 protein. It is used for flow cytometry and immunofluorescence applications to detect and visualize the expression of NFAT1.
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2 protocols using nfat1 af488 d43b1
1
Quantifying Activated T-cell Nuclei
2
Quantifying Activated T-cell Nuclei
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Whole cells were fixed with 4% paraformaldehyde in 1× PBS. Both fixed whole cells and fixed nuclei were permeabilized with 0.3% Triton X-100 in1× PBS and 2% FBS and stained with fluorescent Abs for 15 min at room temperature followed by three washes in 1× PBS and 2% FBS. To sufficiently pellet nuclei, centrifugation was performed at 1000 × g. The following Abs were used for staining: β-tubulin-AF647 (9F3, #3624; Cell Signaling Technology), CD3ε-APC (145–2C11; BD Biosciences), NFAT1-AF488 (D43B1, #14324; Cell Signaling Technology), and NF-κB (p65) (D14E12, #8242; Cell Signaling Technology). Propidium iodide was sourced from Thermo Fisher Scientific. Samples were collected on an LSR 11 flow cytometer (BD Biosciences) and analyzed with FlowJo 10.4.2 (BD Biosciences/Tree Star). After gating on singlets, OT-I nuclei were identified as CellTrace Violethi, β-tubulinlo. At least 10,000 OT-I nuclei events were recorded per sample.
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