facs sorting  (AMS Biotechnology)

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A-B) Generation of HLA-A2 + Luciferase + human oligodendrocytic cell line MO3.13-A2-Luc. (A-B) FACS analysis of the (A) HLA-A2 expression on wildtype (WT) MO3.13 cells (left), MO3.13-A2 cells 2 weeks after transfection with HLA-A2 expression vector (middle), and MO3.13-A2 cells after the FACS sorting for HLA-A2 (right). (B) GFP expression on MO3.13-A2-Luc cells after lentiviral transduction (left), GFP expression on FACS sorted MO3.13-A2-Luc cells (middle) and HLA-A2 expression on MO3.13-A2-Luc cells (right). Grey histograms represent the isotype control and untransduced cells for the analysis of HLA-A2 and GFP expression respectively. (C-E) Optimization of siRNA reverse transfection protocol for MO3.13-A2-Luc cells. (C) Real-time live cell imaging of MO3.13-A2-Luc cells transfected either with a non-targeting siRNA control (Scr) or with a siRNA cocktail targeting genes essential for cell survival (cell death siRNA, siCD). Transfected cells were imaged via the IncuCyte SX5 system for 72 h and real-time % cell confluency was quantified. (D) RT-qPCR analysis of PDL1 mRNA expression in MO3.13-A2-Luc cells transfected either with Scr siRNA or pool of 4 non-overlapping siRNAs targeting PDL1 (siCD274). Results are presented as fold change compared to the Scr after β-actin mRNA normalization. (E) FACS analysis of PD-L1 surface expression on MO3.13-A2-Luc cells transfected either with Scr or PD-L1 specific siRNA pool. Left: representative histograms indicating % PD-L1 expression, right: mean fluorescent intensity (MFI) of PD-L1 from 2 independent FACS data. (F) Phenotypic characterization of FluTC. The expression of CD4 and CD8 was determined on CD3 + FluTC, whereas the expression of effector and memory markers CD45RO and CD62L and co-inhibitory immune checkpoint molecules PD-1, LAG-3 and TIM-3 was determined on CD3 + CD8 + FluTC. (Tn: naïve T cells, Tcm: central memory T cells, Teff: terminal effector T cells, Tem: effector memory T cells). Grey histograms represent the isotype control. (G) Luciferase-based cytotoxicity assay to optimize the co-culture conditions for MO3.13-A2-Luc and FluTC. MO3.13-A2-Luc were pulsed with serial dilutions of the flu-peptide and co-cultured with FluTC. After 20 h of co-culture, remaining relative luciferase units (RLU) were measured. RLU measured in pulsed samples were normalized to RLU of unpulsed (no peptide) control. Statistical significance was calculated compared with no peptide control. (H) Selection of cytotoxicity and viability controls for the HTP screen. MO3.13-A2-Luc cells were transfected with the indicated siRNAs. After 72 h of transfection cells were pulsed with 0,01 µg/ml flu-peptide and co-cultured either with FluTC or treated with control T cell medium (CM). 20 h following co-culture, remaining RLU was measured. The RLU of each sample was normalized to the Scr1 control in the cytotoxicity (FluTC) or viability (CM) setting to determine the impact of gene knockdown. (A-G) Representative data of at least 2 independent experiments. (H) Cumulative data of three independent experiments each performed with four replicates per sample. (D, G-H) Graphs show mean +/- SD. p-values were calculated using two-tailed student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001.

Article Snippet: MO3.13-A2-Luc cells were generated after transduction of MO3.13-A2 cells with lentiviral particles encoding firefly luciferase and GFP (GenTarget Inc – Amsbio, #LVP020 (CMV-Luciferase (firefly)-2A-GFP (Puro)) and FACS sorting.

Techniques: Luciferase, Expressing, Transfection, Plasmid Preparation, Transduction, Control, Live Cell Imaging, Quantitative RT-PCR, Cytotoxicity Assay, Co-Culture Assay, Cell Culture, Selection, Knockdown, Two Tailed Test