XÂY DỰNG QUY TRÌNH NHUỘM MIỄN DỊCH HUỲNH QUANG ĐỂ PHÁT HIỆN OCTN2 TRONG DÒNG TẾ BÀO LYMPHOBLAST NGƯỜI

Abstract

Primary carnitine deficiency (PCD; OMIM #212140) is an autosomal recessive disorder caused by pathogenic variants in SLC22A5, encoding the high-affinity carnitine transporter OCTN2. Accurate detection of OCTN2 expression is crucial for understanding its role in carnitine homeostasis, yet standardized protocols for lymphoblast cell lines (LCLs)—a stable, accessible model—are lacking. Here, we developed and optimized an immunofluorescence (IF) protocol for visualizing OCTN2 in Epstein-Barr virus (EBV)-transformed human LCLs derived from healthy volunteers. Cells were fixed with 4% paraformaldehyde (PFA), permeabilized, blocked, and stained with anti-OCTN2 primary antibodies followed by FITC-conjugated secondary antibodies. Optimization revealed PFA superior to general cytology preservative for preserving antigenicity and morphology. A 60-minute primary antibody incubation yielded maximal signal intensity (mean fluorescence intensity [MFI]: 300 ± 8 arbitrary units [AU], n=3, p<0.001 vs. 30 min), with pre-staining fixation minimizing artifacts. The protocol was reproducible across replicates, showing clear plasma membrane localization with low background, and is compatible with flow cytometry for quantification. This is the first validated IF method for OCTN2 in LCLs, providing a robust tool for studying transporter expression in metabolic research.