Nuclei isolation and flow cytometry staining of OT-I cells stimulated in co-cultures were performed as previously described (20 ). Briefly, CD8+ T cells were negatively enriched from OT-I spleens (Stemcell Technologies) and labeled with CellTrace Violet cell tracking and proliferation dye (Thermo Fisher) for 20m prior to co-culture with peptide-pulsed B6 splenocytes at a 5:1 ratio of APCs to T cells. For nuclei isolation, cells were treated and washed with sucrose and detergent buffers. The nuclei were fixed in 4% paraformaldehyde and then intranuclear staining was performed with a 0.3% Triton-X 100 detergent PBS buffer. NFAT1-AF488 antibody (clone D43B1) was purchased from Cell Signaling Technology (Danvers, MA) and NFAT2-PE antibody (clone 7A6) was purchased from BioLegend (San Diego, CA). The PrimeFlow assay kit, from Thermo Fisher, was used for RNA flow. Cd69 and Irf4 probe sets were ordered for AF647.
Primeflow assay kit
Manufactured by Thermo Fisher Scientific
The PrimeFlow assay kit is a flow cytometry-based tool designed for the simultaneous analysis of gene expression and protein levels within individual cells. The kit utilizes branched DNA technology to amplify and detect target mRNA sequences, allowing for the quantification of gene expression along with the measurement of surface or intracellular proteins.
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Lab products found in correlation
2 protocols using primeflow assay kit
1
Isolation and Analysis of Activated OT-I T Cells
2
Flow Cytometric Analysis of Engineered Cells
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Seven days after electroporation, cells were subjected to prime flow analysis following the manufacturer’s instruction kit (Prime Flow assay kit, Thermo Fisher Scientific, cat. no. 8-18005-210). The probes used are indicated in Table S4 . ΔLNGFR and viability staining (FVD450, eBioscience cat. no. 65-0863-13) were carried out during the procedure. Expression was analyzed on a flow cytometer.
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