Primeflow rna assay

PrimeFlow RNA assay, a flow cytometry based RNA detection technology, was conducted following manufacturer’s instructions (eBioscience, San Diego, CA). Specifically, 1 × 10⁶ cells were harvested, permeabilized followed by intracellular staining using PE anti-human CD79a (clone: HM47). Cells were then incubated with EBF1 mRNA specific target probes (VA1-19733; Affymetrix, Santa Clara, CA) for 2 hours. After incubation, cells were washed and incubated with pre-Amplification and Amplification probes for 3 h, followed by incubation with fluorophore conjugated label probes for 1 h. Cells were resuspended in FACS buffer (1X HBSS containing 1% BSA and 0.1% sodium azide, pH 7.4–7.6) and analyzed by flow cytometry.

Skin tissue single-cell suspensions were analyzed for mRNA and protein expression using the PrimeFlow RNA assay (eBioscience) and standard mouse probesets for Ifng, Il5, Il13, and Il17a, as per manufacturer’s instructions for 96-well-plate staining.

PrimeFlow™ RNA assay (eBiosciences©, San Diego, CA) was performed per manufacturer’s directions. Treated PBMCs were fixed, permeabilized, and bound with IFNΑ2 probe. The mRNA signal was then amplified and detected using Alexa Fluor 647 detection probes (Thermo-Fisher Scientific, Waltham, MA). Relative gene expression was determined via flow cytometry.

Freshly isolated human PBMCs were stimulated with SAgs for 4 h, unless otherwise stated, in the presence of 1 μM brefeldin A (Sigma-Aldrich). Cells were thoroughly washed and stained at 4°C with fluorochrome-conjugated mAbs to cell surface molecules (Supplemental Table II). For intracellular detection of cytokines, the Intracellular Fixation and Permeabilization Buffer Set from eBioscience was used. We also employed a Mouse IL-17A Secretion Assay kit from Miltenyi Biotec (Auburn, CA) to confirm the secretion of this cytokine. For detection of transcription factors, the FoxP3 Staining Buffer Set (eBioscience) was used. For RNA staining, the Prime FlowRNA Assay (eBioscience) was used with probes designed for the detection of IL-17A, IFN-γ and RPL13A signals. Cells were treated and stained for surface and intracellular molecules (Supplemental Table II) following the manufacturer’s instructions. Isotype and/or fluorescence minus one controls were used in parallel for proper gating. Samples were run on a BD FACSCanto II or a BD LSR II (when using violet fluorochrome-conjugated mAbs) and analyzed by FlowJo software (Tree Star).