Quantifying T-cell Differentiation Markers

Total RNA was isolated using Trizol Reagent (Thermo Fisher, Waltham, MA, USA) according to manufacturer instructions. RNA quantity and purity were measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Sample purity was checked at A260/A280 ratio between 1.80 and 2.00. Gene expression was measured by RT-qPCR by using validated primers for T-bet, RORγT, GATA3, FOXP3, IL-10, IL-21 and TGFβ after 7 days from addition of SULF A to MLR test. 18S Ribosomal RNA (rRNA) was used as a housekeeping gene to normalize sample-to-sample systematic variation in RT-qPCR. ΔCt method was used to calculate the relative gene expression.

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Barra G., Gallo C., Carbone D., Ziaco M., Dell’Isola M., Affuso M., Manzo E., Nuzzo G., Fioretto L., D’Ippolito G., De Palma R, & Fontana A. (2023). The immunoregulatory effect of the TREM2-agonist Sulfavant A in human allogeneic mixed lymphocyte reaction. Frontiers in Immunology, 14, 1050113.

Publication 2023

Trizol Reagent NanoDrop 2000 spectrophotometer

18s ribosomal rna Gata3 Gene expression Housekeeping gene Il 10 Il 21 Mlr test Primers Rorγt Tgfβ Trizol

Corresponding Organization : University of Naples Federico II

Other organizations : University of Genoa

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Variable analysis

independent variables

Addition of SULF A to MLR test

dependent variables

Gene expression of T-bet, RORγT, GATA3, FOXP3, IL-10, IL-21, and TGFβ

control variables

RNA quantity and purity were measured by a NanoDrop 2000 spectrophotometer
Sample purity was checked at A260/A280 ratio between 1.80 and 2.00
18S Ribosomal RNA (rRNA) was used as a housekeeping gene to normalize sample-to-sample systematic variation in RT-qPCR

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