cell line present  (ATCC)

Journal: Frontiers in Aging Neuroscience

Article Title: Urolithin A and nicotinamide riboside differentially regulate innate immune defenses and metabolism in human microglial cells

doi: 10.3389/fnagi.2024.1503336

Figure Lengend Snippet: Treatment with UA and NR promotes a significant increase in maximal and spare capacity OCR in HCM3 cells, with maximal glycolytic function increased only by UA treatment. HCM3 cells (2.2 × 10 4 cells/well) untreated (Ctr.) or treated with 10 μM UA or 3 mM NR for 1 week were incubated in appropriate medium containing 2 mM glutamine and 11 mM glucose as metabolic energy substrates for OCR measurement and 2 mM glutamine for PPR measurement. (A, B) show representative traces of oxygen consumption rates (OCR). Oligomycin (Oligo, 1 μg/mL), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 200 nM each addition), and rotenone (Rot, 1 μM) plus antimycin A (AA, 1 μM) were added where indicated. Different OCR parameters were determined: (C) Maximal respiratory capacity and (D) Spare respiratory capacity. For evaluation of glycolytic function, Panels E and F represent the experimental design and raw data of OCR and extracellular acidification rate (ECAR). Glucose (10 mM), rotenone (Rot, 1 μM) plus antimycin A (AA, 1 μM), and monensin (100 μM) plus FCCP (1 μM) were added where indicated. (G) Basal glycolytic proton production (PPRglyc) and maximal glycolytic rate stimulated by rotenone plus antimycin A (H) and monensin plus FCCP (I) were calculated from OCR and ECAR. Each dot represents an independent experiment, and bars denote means (+ SEM) ( N = 4). ** P ≤ 0.01 vs. untreated cells, Ctr.; * P ≤ 0.05 vs. untreated cells, Ctr. # P ≤ 0.05 vs. NR-treated cells. (J) HMC3 cells were treated with DMSO, 10 μM UA, or 3 mM NR for 6 days, respectively. The FCCP group was treated with 20 μM FCCP for 10 min and controlled for by using DMSO. Data was presented as mean with standard deprivation. Data were statistically tested using ordinary one-way ANOVA by Prism 10. *** P < 0.001.

Article Snippet: The HMC3 cell line present in this study was obtained from ATCC (CRL-3304) and was cultured in Minimum Essential Medium Eagle (Merck) containing 10% fetal bovine serum and 1% GlutaMAX (Thermo Fisher Scientific).

Techniques: Incubation

Journal: Frontiers in Aging Neuroscience

Article Title: Urolithin A and nicotinamide riboside differentially regulate innate immune defenses and metabolism in human microglial cells

doi: 10.3389/fnagi.2024.1503336

Figure Lengend Snippet: UA and NR differentially regulate transcription of several genes in microglia cells. (A) NanoString analysis was done to study neuroinflammation-related genes. Heatmap showing the directed global significance scores: orange denotes gene sets whose genes exhibit increased differential expression with the covariate, blue denotes gene sets with less differential expression. (B–E) Individual NanoString pathway scores following UA, NR, and/or DNA stimulation of HMC3 cells. (F) Venn diagram comparing the differentially expressed genes of UA vs. controls and NR vs. controls. There were ten genes that were in common, 111 UA subgroup-specific genes and 13 NR subgroup-specific genes. (G, H) Volcano plot representation of differential gene expression analysis of UA vs. controls and NR vs. controls. (I–L) Representative western blot analysis showing changed level of the TREM2, RIG-I, STING, and p21 following UA and/or NR treatment of HMC3 cells compared to control (UTR), analysed by two-way ANOVA. Each dot represents an independent experiment and bars denote means (+ SEM) ( N = 4). ** P ≤ 0.01 vs . untreated cells, Ctr.; * P ≤ 0.05 vs . untreated cells, Ctr. *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: The HMC3 cell line present in this study was obtained from ATCC (CRL-3304) and was cultured in Minimum Essential Medium Eagle (Merck) containing 10% fetal bovine serum and 1% GlutaMAX (Thermo Fisher Scientific).

Techniques: Quantitative Proteomics, Gene Expression, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: In Vitro Validation of a Genetically Engineered T-Cell Activation Model. (A) Schematic representation of the genetically modified Jurkat T-cell line designed to report T-cell activation via NFAT-driven eGFP expression. These cells are equipped with a TCR specific for HLA-A*02-restricted MART1, CD8 co-receptor, PD-1 receptor, and a dCas9-KRAB-MeCP2 transcriptional repression system. When co-cultured with APCs/melanoma cells expressing MART1, eGFP expression indicates robust T-cell activation. In certain melanoma cell lines, robust expression of surface PD-L1 can be achieved by pre-treatment for 24 hours with IFN-γ. (B) Validation of the NFAT-driven eGFP reporter functionality in the T-cell model using flow cytometry. Negligible T-cell activation is observed in the absence of antigen-presenting or tumor cells (quiescent state), while treatment with PMA and ionomycin results in near-total activation. (C) Comparative analysis of T-cell activation following co-culture with different antigen-presenting cells (APCs) pulsed with MART1 peptides and melanoma cell lines. 2D3s were co-cultured at a 1:2 tumor cell/APC to 2D3 TCR/dCas9 ratio for 20-24 hours. The 2D3 TCR/dCas9 T-cell line exhibits moderate activation with peptide-pulsed U266-B1 cells and pronounced activation with peptide-pulsed T2 cells. Negative controls with non-peptide-pulsed APCs confirm assay specificity. As for melanoma cell lines, additional examination of checkpoint blockade with nivolumab and IFN-γ treatment effects on co-culture-mediated T-cell activation demonstrates distinct immune evasion mechanisms. All data shown represent the mean values of triplicate measurements, with error bars indicating the standard deviation.

Article Snippet: The embryonic kidney cell line HEK293T/17 (ATCC, Cat. #CRL-11268), the malignant melanoma cell lines MALME-3M (ATCC, Cat. #HTB64) and SK-MEL-5 (ATCC, Cat. #HTB-70), the 2D3 cell line, a Jurkat derivative, and antigen presenting cell lines (APCs) T2 (174 x CEM.T2, ATCC, Cat. #CRL-1992), and U266B1 (ATCC, Cat. #TIB-196) were employed in the study.

Techniques: In Vitro, Activation Assay, Genetically Modified, Expressing, Cell Culture, Flow Cytometry, Co-Culture Assay, Standard Deviation