celltox tm green cytotoxicity assay kit g8741  (Promega)

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B, CAOV3, TYKNU, TOV21G, and TOV112D cells were treated with ( A ) 10µM erastin or ( B ) 250nM RSL3 in the presence or absence of 2-8% ascites from three OVCA patients. Liproxtasin-1 (Lipro-1) (2µM) was used as a control ( n =3/cell line, 24 hours). C-D , cells were treated with 5µM erastin for 20 hours or 2µM RSL3 for 2 hours in the presence or absence of 2% ascites. Lipid peroxidation was measured via flow cytometry analysis of BODIPY TM 581/591 C11 staining ( n provided in panel). E-F , cells were either pre-treated with 8% OVCA ascites (Day 0, 16 hours) or left untreated and subsequently treated with 10µM erastin and 8% OVCA ascites. Cell death was visualized ( E ) and measured ( F ) via the CellTox TM Green assay (Days 2-4) in which green fluorescence indicates cell death ( n =3, scale=80µm). G , cells were treated with 10µM erastin in the presence or absence of 2-8% ascites from liver cirrhosis (LC) or OVCA patients for 24 hours ( n provided in panel, 24 hours). H , micro-organospheres (MOS) were treated with 50µM IKE in the presence or absence of 10-40% OVCA ascites for 72 hours ( n =5). I-J , CAOV3 cells transduced with lentivirus expressing luciferase were resuspended in PBS or human OVCA ascites and IP injected into 6-week-old SCID beige mice. Tumor growth ( I ) was visualized and ( J ) measured as total photon flux (p/s) 10 days after tumor injection via IVIS bioluminescent imaging ( n =6). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using correlated-samples one-way or two-way ( F ) ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method ( D-H ). Statistical significance for J was assessed using the two-tailed Student’s t -test method.

Article Snippet: The CellTox TM Green cytotoxicity assay (Promega, G8741) was used for the visualization and measurement of cell death at multiple timepoints.

Techniques: Control, Flow Cytometry, Staining, CellTox Assay, Fluorescence, Transduction, Expressing, Luciferase, Injection, Imaging, Glo Assay, Two Tailed Test

Journal: Cancers

Article Title: ADAM17 Inhibition Increases the Impact of Cisplatin Treatment in Ovarian Cancer Spheroids

doi: 10.3390/cancers13092039

Figure Lengend Snippet: Multi-content 3D readout to test drug combinations. OvCa cells were seeded in ultra-low attachment (ULA) plates and spheroids formed four days before treatment. Daily imaging using NYONE ® Scientific (SYNENTEC) recorded the formation process and spheroid morphology and growth over time. On day four, spheroids were treated with cisplatin (asterisks) in absence or presence of metalloprotease inhibitors (blue circles), as described. To allow multiplex readout of three consecutive assays in one 96-well plate, the real-time fluorescence-based cytotoxicity assay CellTox TM Green was first applied on day five and imaged twice: day five = 24 h after treatment, and day six = 48 h after treatment. Next, cell viability and caspase activation were measured using two different luminescent assays to allow for multiplexing using different filters.

Article Snippet: The next day, CellTox TM Green cytotoxicity (Promega #G8743) stain was added in a final dilution of 1:2000 and imaged 3 h later using NYONE ® Scientific and the following settings: brightfield (Ex: BF, Em: Green (530/43 nm)), CellTox TM Green (Ex: Blue (475/28 nm); Em: Green (530/43 nm)).

Techniques: Imaging, Multiplex Assay, Fluorescence, Cytotoxicity Assay, CellTox Assay, Activation Assay, Multiplexing

Journal: Cancers

Article Title: ADAM17 Inhibition Increases the Impact of Cisplatin Treatment in Ovarian Cancer Spheroids

doi: 10.3390/cancers13092039

Figure Lengend Snippet: Combined cytotoxic effects of GW280264X and cisplatin in OvCa cell line spheroids can be visualized and quantified using automated imaging. OvCa cell lines were cultured for 4 days in ULA plates for formation of spheroids and treated with the indicated concentrations of cisplatin (Cis) and NaCl. ADAM17 activity was inhibited using GW280264X (GW) and ADAM10 by GI254023X (GI). DMSO was used as a solvent control. At 48 h after treatment, the spheroids were imaged using NYONE ® Scientific. ( a ) Representative brightfield (BF) images 48 h after treatment. Displayed are concentrations achieving the strongest combinatorial effects (HEY: 5 µM cisplatin; SKOV-3: 40 µM cisplatin, OVCAR-8: 10 µM cisplatin.). ( b ) Corresponding CellTox TM Green (CTG) images. Orange line indicates spheroid area as determined in the BF image by the spheroid quantification (1F) application of YT ® -Software. The strongest CTG staining was observed with the combination of cisplatin and GW280264X (lower panel, right image). Objective: 4×, Scale: 250 µm. ( c ) Normalized CTG intensities of at least three biological replicates demonstrate the combined effect of GW280264X and cisplatin. The mean ± SEM is displayed. Curve fitting was performed using GraphPad Prism 9. Based on the Shapiro–Wilk normality test, EC 50 values of biological replicates were analyzed by ANOVA following Tukey´s multiple comparison test. ( d ) EC 50 values of CTG; ( e ) drug reduction index (DRI 50 ) at 50% effectiveness. Synergistic effect (DRI 50 < 1), additive effect (DRI 50 = 1), and antagonistic effect (DRI 50 > 1). ** p < 0.01, **** p < 0.0001.

Article Snippet: The next day, CellTox TM Green cytotoxicity (Promega #G8743) stain was added in a final dilution of 1:2000 and imaged 3 h later using NYONE ® Scientific and the following settings: brightfield (Ex: BF, Em: Green (530/43 nm)), CellTox TM Green (Ex: Blue (475/28 nm); Em: Green (530/43 nm)).

Techniques: Imaging, Cell Culture, Activity Assay, Solvent, Control, CellTox Assay, Software, Staining, Comparison

Journal: Cancers

Article Title: ADAM17 Inhibition Increases the Impact of Cisplatin Treatment in Ovarian Cancer Spheroids

doi: 10.3390/cancers13092039

Figure Lengend Snippet: Translational application of multiplex workflow confirms the combinatorial effect of GW280264X and cisplatin in primary OvCa spheroids. Primary cells were isolated from tumor and ascites of three OvCa patients, seeded in ULA plates, and treated after four days of spheroid formation. ( a ) Caspase 3/7 activation 48 h after treatment revealed combinatorial effects by cisplatin treatment (Cis) and inhibition of ADAM17 with GW280264X (GW) compared with sole inhibition of ADAM10 by GI254023X (GI) in tumor-derived cells. DMSO was used as the solvent control. This effect was even more pronounced in ascites. ( b ) CellTox TM Green staining confirmed these combinatorial effects. Representative images show the best combined effect using 20 µM cisplatin (cis). Upper panel: brightfield; Lower panel: CellTox TM Green. Orange line indicates spheroid area. Magnification 4×. Scale: 250 µm. Data are presented as mean + SD of three technical replicates. As for restricted patient material, no statistical analysis was performed using technical replicates.

Article Snippet: The next day, CellTox TM Green cytotoxicity (Promega #G8743) stain was added in a final dilution of 1:2000 and imaged 3 h later using NYONE ® Scientific and the following settings: brightfield (Ex: BF, Em: Green (530/43 nm)), CellTox TM Green (Ex: Blue (475/28 nm); Em: Green (530/43 nm)).

Techniques: Multiplex Assay, Isolation, Activation Assay, Inhibition, Derivative Assay, Solvent, Control, CellTox Assay, Staining