alkaline phosphatase seap reporter assay raw blue cells  (InvivoGen)

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