reporter cell lines  (Fisher Scientific)

Journal: Marine Drugs

Article Title: From Sea to Cell: Ascophyllum nodosum and Fucus vesiculosus Extracts Attenuate NF-κB-Mediated Inflammation and Protect Intestinal Barrier Integrity—A Comprehensive Analysis Applying In Vitro and In Vivo Models

doi: 10.3390/md24050182

Figure Lengend Snippet: Effects of AN and FV on NF-κB activation in reporter cell lines. NF-κB activation was determined in ( A ) non-TLR4-expressing HEK-Blue null2 cells and ( B ) TLR4-expressing HEK-Blue hTLR4 cells following 24 h of stimulation with TNF-α (10 ng/mL) or LPS (10 ng/mL) and AN or FV (200 µg/g). NF-κB activity was quantified by SEAP levels in the culture supernatant and normalized to the respective stressor control. Statistical analysis: one-way ANOVA with Tukey’s multiple comparison test. Data are presented as median with interquartile range (IQR), whiskers (min/max values), and individual data points from three independent experiments ( n = 9). Significant p values are indicated as ** ( p ≤ 0.01), *** ( p ≤ 0.001), **** ( p ≤ 0.0001).

Article Snippet: The HEK-Blue hTLR4 reporter cell line, which constitutively expresses the human toll-like receptor 4 (TLR4) together with an NF-κB-responsive secreted embryonic alkaline phosphatase (SEAP) reporter, and its corresponding control cell line HEK-Blue null2 (expressing SEAP only) were obtained from InvivoGen (San Diego, CA, USA).

Techniques: Activation Assay, Expressing, Activity Assay, Control, Comparison

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Estradiol dose-response and recovery of spiked estradiol from tampon extracts. (A) T47D-luc cells were treated with an 11-point estradiol dilution series starting at 30nM, followed by 10nM, 3.3nM, 1.11nM, 370pM, 123pM, 41pM, 14pM, 4.6pM, 1.5pM, and 0.5pM). For each concentration, 2 μL of steroid dilution was added to 198 μL assay medium, and cells were incubated for 24 hours. Estrogen-responsive luciferase activity was quantified using BrightGlo substrate. Luminescence values represent the mean of 4 technical replicates, and a sigmoidal dose-response curve with EC 50 determination was generated using GraphPad v10. (B) Recovery of estradiol from tampon brands A-D following spiking and extraction. One gram of each tampon from Brands A, B, C, and D were spiked with 2nM estradiol, soaked in a 1:1 water:ethanol solution for 4 hours, extracted, evaporated to dryness, and reconstituted in methanol. Extracts were diluted 1:100 in cell culture medium and then serially diluted 1:2 to generate 4 assay points. T47D-luc cells were incubated with a further 1:10 dilution of each tampon extract for 24 hours and luciferase activity measured using BrightGlo. Each point represents mean ± SEM of 4 technical replicates corrected for the blank control sample (1:1 ethanol:water solution SPE-extracted as per samples). The data for the spiked tampons are plotted alongside estradiol standards run in parallel.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Concentration Assay, Incubation, Luciferase, Activity Assay, Generated, Extraction, Cell Culture, Control

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Estrogenic activity detected in extracts from 8 commercially available tampon brands (A-H). T47D-luc cells were exposed to methanolic extracts prepared from 1 g of core tampon material (string removed) from 8 tampon brands sold in New Zealand. Each 1 g sample was soaked in a 1:1 ethanol:water solution for 4 hours, sonicated, filtered, extracted, and then evaporated to dryness, and reconstituted in methanol. The final extract was diluted into assay medium at 1:1000 (1), followed by serial dilutions to generate 1:2000 (2), 1:4000 (3), and 1:8000 (4) exposure concentrations. Cells were incubated with each dilution for 24 hours, and estrogen-responsive luciferase activity was quantified using a luminescence plate reader. Data are presented as the mean ± SEM of 2 independently extracted tampons per brand, each tested as 2 portions of 1 g each, and each dilution tested with 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Activity Assay, Sonication, Incubation, Luciferase, Microplate Reader Luminescence Measurement

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Independent batches of Tampon Brand D show estrogenic activity. T47D-luc cells were treated with methanolic extracts generated from 1 g of core tampon material (string removed) from 3 independent batches of Brand A and Brand D. For each batch, 3 separate tampons were extracted independently. Each 1 g sample was soaked in 1:1 ethanol:water solution for 4 hours, sonicated, filtered, extracted, and evaporated to dryness, and reconstituted in methanol. Reconstituted extracts were diluted into assay medium at 1:1000 (1), followed by serial 1:2 dilutions to produce 1:2000 (2), 1:4000 (3), and 1:8000 (4) exposure concentrations. Cells were incubated for 24 hours, and estrogen-responsive luciferase activity was quantified by using a luminescence plate reader. Data are shown as mean ± SEM of 3 independently extracted tampons per batch, tested as 2 portions of 1 g each with each dilution tested as 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Activity Assay, Generated, Sonication, Incubation, Luciferase, Microplate Reader Luminescence Measurement

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Estrogenic activity of internationally sourced tampon brands. T47D-luc cells were treated with methanolic extracts prepared from intact tampons (string removed) from a panel of internationally sourced tampon brands (Brands I-R). For each brand, 2 or 3 independent tampons were extracted separately on independent days. Each tampon was soaked in a 1:1 ethanol:water solution for 4 hours, sonicated, filtered, extracted, evaporated to dryness, and reconstituted in methanol. Reconstituted extracts were diluted into assay medium at 1:1000, and cells were incubated for 24 hours before estrogen-responsive luciferase activity was quantified using a luminescence plate reader. Data are shown as mean ± SEM of 2 or 3 independently extracted tampons per brand with each dilution tested as 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Activity Assay, Sonication, Incubation, Luciferase, Microplate Reader Luminescence Measurement

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Effect of sonication on estrogenic activity of tampon extracts. T47D-luc cells were exposed to methanolic extracts prepared from 2 independent tampons per brand (Brands A and D, string removed), comparing extraction with and without sonication. Each tampon was soaked in a 1:1 ethanol:water solution for 4 hours, either sonicated or left nonsonicated, then filtered, extracted, evaporated to dryness, and reconstituted in methanol. Extracts were diluted into assay medium at 1:1000 (1), followed by serial 1:2 dilutions to generate 1:2000 (2) and 1:4000 (3) exposure concentrations. Cells were incubated for 24 hours, and estrogen-responsive luciferase activity was quantified. Data represent mean ± SEM of t2wo independently extracted tampons per condition with each dilution tested as 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Sonication, Activity Assay, Extraction, Incubation, Luciferase

Journal: bioRxiv

Article Title: Discovery and Preclinical Validation of a Clinically Optimized Mitochondrial Complex I Modulator for Alzheimer’s Disease

doi: 10.64898/2026.04.10.717554

Figure Lengend Snippet: a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter HEK293 cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.

Article Snippet: NF-κB Reporter (Luc) HEK293 cell line and the antioxidant response element (ARE) Luciferase Reporter HepG2 hepatic cell line were purchased from BPS Bioscience (CA, USA) and were cultured in accordance of manufacturer instructions.

Techniques: Expressing, Activation Assay, Luciferase, Activity Assay, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Discovery and Preclinical Validation of a Clinically Optimized Mitochondrial Complex I Modulator for Alzheimer’s Disease

doi: 10.64898/2026.04.10.717554

Figure Lengend Snippet: a A drug discovery funnel used for the identification of C273. b C273 protects MC65 Tet-Off cells from Aβ toxicity (red line). # indicates cell death in Tet-Off cells treated with vehicle. No toxicity was observed in MC65 Tet-On cells (Aβ not expressed, black line). EC 50 values were calculated using nonlinear regression in GraphPad Prism 10. c Treatment with C273 for 24 h does not induce cytotoxicity in HepG2 human hepatic cells at concentrations up to 100 μM. d C273 increases NADH levels in MC65 Tet-On cells, consistent with inhibition of mtCI. NADH levels were measured over 24 h. e Inhibition of mtCI activity in isolated mouse brain mitochondria by C273 measured using an NADH oxidation assay. f Activities of mitochondrial ETC complexes I–IV were assessed in permeabilized MC65 cells using an electron flow assay in the Seahorse XFe96 Extracellular Flux Analyzer in the presence of C273 (25 and 50 μM). g Injection of rotenone or C273 to monitor OCR kinetics in MC65 Tet-On cells. h Pretreatment with rotenone blocks the protective effect of C273 in MC65 Tet-Off cells (Aβ expressed). Open orange squares: Tet-On cells, no Aβ expression, treated with vehicle (positive control, 100% viability). Blue triangles: Tet-Off cells (Aβ expressed), which undergo extensive cell death after 3 days. Tet-Off cells treated with C273 (2 nM - 5 μM; blue line) show complete protection from Aβ toxicity. Solid red triangles: Tet-On cells treated with 32 nM rotenone, which show no toxicity. Open red triangles: Tet-Off cells treated with 32 nM rotenone alone, resulting in complete cell death. Pretreatment with 32 nM rotenone blocks the protective effect of C273 in Tet-Off cells (red line, grey box), supporting a competitive interaction at mtCI. Partial rescue at higher C273 concentrations reflects incomplete occupancy of the mtCI binding site by rotenone, which inhibits ∼50% of mtCI activity at this dose. The grey box indicates the EC 50 concentration range for C273. P values were calculated using unpaired two-tailed Student’s t -tests; ** P < 0.01. Data are presented as mean ± SD ( n = 4 technical replicates). All experiments were replicated at least twice.

Article Snippet: NF-κB Reporter (Luc) HEK293 cell line and the antioxidant response element (ARE) Luciferase Reporter HepG2 hepatic cell line were purchased from BPS Bioscience (CA, USA) and were cultured in accordance of manufacturer instructions.

Techniques: Drug discovery, Inhibition, Activity Assay, Isolation, Oxidation Assay, Injection, Expressing, Positive Control, Binding Assay, Concentration Assay, Two Tailed Test