High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis.

High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis. J Immunol Methods. 2019 Nov 11;:112667 Authors: Tsai WL, Vian L, Giudice V, Kieltyka J, Liu C, Fonseca V, Gazaniga N, Gao S, Kajigaya S, Young NS, Biancotto A, Gadina M Abstract Fluorescent cell barcoding (FCB) is a multiplexing technique for high-throughput flow cytometry (FCM). Although powerful in minimizing staining variability, it remains a subjective FCM technique because of inter-operator variability and differences in data analysis. FCB was implemented by combining two-dye barcoding (DyLight 350 plus Pacific Orange) with five-color surface marker antibody and intracellular staining for phosphoprotein signaling analysis. We proposed a robust method to measure intra- and inter-assay variability of FCB in T/B cells and monocytes by combining range and ratio of variability to standard statistical analyses. Data analysis was carried out by conventional and semi-automated workflows and built with R software. Results obtained from both analyses were compared to assess feasibility and reproducibility of FCB data analysis by machine-learning methods. Our results showed efficient FCB using DyLight 350 and Pacific Orange at concentrations of 0, 15 or 30, and 250 μg/mL, and a high reproducibility of FCB in combination with surface marker and intracellular antibodies. Inter-operator variability was minimized by adding an internal control...
Source: Journal of Immunological Methods - Category: Allergy & Immunology Authors: Tags: J Immunol Methods Source Type: research