To assess the effect of ascites on proliferation, 5,000 Colo-205 and SNU-C1 cells/well were seeded in 96-well plate in triplicate and were grown in serum-free medium (SFM) supplemented with various concentration of cell-free ascites (CFA) as indicated. Cell proliferation was assessed at day 0 and day 5 using CellTitre-Glo assay (Promega) following the manufacturer’s instructions. For in vitro drug treatment, 5,000 Colo-205 cells/well were seeded in 96-well plate in triplicate and were grown in SFM supplemented with 5% CFA (5% CFA medium) or SFM supplemented with 10% FBS (10% FBS medium) overnight, followed by treatment with 11 concentrations of various inhibitors (PAI-1 inhibitors: TM5441 and Tiplaxtinin, STAT3 inhibitor: Napabucasin, PI3K/mTOR inhibitor: BEZ235, DNA crosslinker: Mitomycin C) and DMSO vehicle for 72 hours. Cell viability after drug treatment was determined using CellTitre-Glo assay (Promega).
Celltitre glo assay
Manufactured by Promega
Sourced in United States
The CellTiter-Glo assay is a luminescent cell viability assay that quantifies the amount of ATP present, which is an indicator of metabolically active cells. The assay involves the addition of a single reagent that results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present.
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Lab products found in correlation
Cisplatin, by Bio-Techne (1 mentions)
Cisplatin, by Promega (1 mentions)
Etoposide, by Promega (1 mentions)
Cell titre glo kit, by Promega (1 mentions)
Etoposide, by Merck Group (1 mentions)
Biotek synergy h1 plate reader, by Agilent Technologies (1 mentions)
White 96 well plate, by PerkinElmer (1 mentions)
Wallac 1420 victor plate reader, by PerkinElmer (1 mentions)
Williams e medium, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Cytotoxicity detection kit, by Roche (1 mentions)
Lncap, by Thermo Fisher Scientific (1 mentions)
Charcoal stripped serum, by Thermo Fisher Scientific (1 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Enzalutamide, by Selleck Chemicals (1 mentions)
Rpmi 1640 glutamax, by Thermo Fisher Scientific (1 mentions)
Peg400, by Merck Group (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Labrasol, by Gattefosse (1 mentions)
29 protocols using celltitre glo assay
1
Ascites Impact on Cell Proliferation
2
Evaluating Cell Viability and DNA Damage
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Cells were cultured at 5,000 cells/well in a 96‐well plate in triplicate for 24 hr, followed by treatment with varying doses of free or encapsulated drugs alone or in combination. Cell viability was determined at 96 hr using the CellTitre Glo assay (Promega) on a Tecan microplate reader. Immunofluorescence was performed as previously described.75 Briefly, to detect HR, COV362, OVCAR4, and OVCAR8 cells were plated at 6.0 × 104 cells on 18‐mm PLL‐coated glass cover sides in 12‐well plates for 24 hr and then treated with 10 μM free or encapsulated drugs for 24 hr. The cells were costained with anti‐rabbit RAD51 (H‐92, sc‐8349, Santa Cruz Biotechnology, TX) and anti‐mouse phosphorylated γH2AX (Ser139), (#JBW301, Millipore, CA) primary antibodies overnight at 4°C. Protein expression was detected with anti‐rabbit IgG Alexa Fluor 647 and anti‐mouse IgG Alexa Fluor 488 (A‐21121, Invitrogen) secondary antibodies with DAPI (0.1 mg/mL) nuclear counterstaining for 2 hr at ambient temperature. Images were acquired on an Applied Precision Delta Vision Microscope (Olympus).
3
Evaluating Chemotherapy Efficacy and Relapse in SCLC
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For the chemotherapy treatment, Cisplatin (Tocris, 5B/266434) and Etoposide (Sigma, 099M4892V) were used. The drugs were added to the wells containing 7-day old SCLC tumors at a concentration equal to their respective half-maximal inhibitory concentration (IC50). To calculate the IC50, the tumors were treated with Cisplatin and Etoposide, singly and in combination, in a range of 0–100 µM, for 72 h. Upon reaching the end point, cell viability was determined via Cell-Titre-Glo assay (Promega) according to manufacturer’s protocol for viability determination. From the concentration and corresponding viability values, IC50 was calculated using Graphpad Prism (version 9) software. All treatments were done in triplicate.
To study cancer relapse after chemotherapy treatment, we measured cell viability by image analysis and using Cell-Titre-Glo kit (Promega) to detect metabolically active cells. All readings were taken in triplicate at 7-day intervals for a duration of 0–38 post-drug treatment days.
To study cancer relapse after chemotherapy treatment, we measured cell viability by image analysis and using Cell-Titre-Glo kit (Promega) to detect metabolically active cells. All readings were taken in triplicate at 7-day intervals for a duration of 0–38 post-drug treatment days.
4
Evaluating Glioma Cell Viability
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Optimized number of U87MG, A172, U118, T98G and E98 cells (3 × 103 to 5 × 103) were seeded in white 96-well plates (Perkin Elmer) and allowed to attach. After 24 h, cells were treated with determined concentration of chemical compounds for 24–72 h. Cell viability was measured using a CellTitre Glo assay (Promega) according the manufacturer’s instructions. Luminescence was read with BioTek Synergy H1 plate reader (BioTek, Country). Bioluminescence was normalized and presented as a per cent of the control.
Cell viability for glioma stem cells was seeded into 96-well plates 5 × 103 cells/well and allowed to attach overnight. Next day medium was changed into a medium containing drugs. After 72 h of treatment medium was changed into fresh NSC+/+ containing 10% of Alamar blue solution (10099022, Fisher Scientific). Fluorescent signal was measured after 210 min of incubation in 37°C using Wallac Victor 1420 plate reader (Perkin Elmer). Fluorescence was normalized and presented as a per cent of the control.
Cell viability for glioma stem cells was seeded into 96-well plates 5 × 103 cells/well and allowed to attach overnight. Next day medium was changed into a medium containing drugs. After 72 h of treatment medium was changed into fresh NSC+/+ containing 10% of Alamar blue solution (10099022, Fisher Scientific). Fluorescent signal was measured after 210 min of incubation in 37°C using Wallac Victor 1420 plate reader (Perkin Elmer). Fluorescence was normalized and presented as a per cent of the control.
5
Cell Viability Assessment Techniques
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Cell viability was assessed using Cell Titre-Glo assay (Promega, USA) and Water-Soluble Tetrazolium (WST)-1 assay (Beyotime, China) according to the manufacturer’s protocols (Promega and Beyotime). All experiments were conducted in triplicate.
6
Cryopreserved Hepatocytes Viability Assay
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Cryopreserved primary human hepatocytes were purchased form BioreclamationIVT (Brussels, Belgium), who obtains and distributes consented human material from a network of institutional review board (IRB) approved collection sites under adherence to effective ethical and regulatory guidelines. The cells were thawed according to manufacturer’s instructions, and cultured at a density of 35’000 cells/well in 96-well BD BioCoat Collagen 1 plates (Becton Dickinson, Bedford, MA). Cells were exposed to test compounds or vehicle (0.1% DMSO) after over-night pre-culture in William’s Medium E (Sigma-Aldrich, Buchs, Switzerland) supplemented with Penicillin/Streptomycin (Life Technologies, Zug, Switzerland).
Cell were harvested 3 h after test compound addition by removing the culture medium. Cell lysates in 50 μl RLT buffer (QIAGEN, Hombrechtikon, Switzerland) were immediately frozen and stored at −80°C.
Intracelluar ATP content was assessed 24 h after test compound addition using the CellTitre-Glo assay (Promega, Dübendorf, Switzerland).
Cell integrity was assessed by the release of the intracellular enzyme lactate dehydrogenase (LDH) into the cell culture supernatant 24 h after test compound addition using the Cytotoxicity Detection Kit (Roche Diagnostics, Rotkreuz, Switzerland).
Cell were harvested 3 h after test compound addition by removing the culture medium. Cell lysates in 50 μl RLT buffer (QIAGEN, Hombrechtikon, Switzerland) were immediately frozen and stored at −80°C.
Intracelluar ATP content was assessed 24 h after test compound addition using the CellTitre-Glo assay (Promega, Dübendorf, Switzerland).
Cell integrity was assessed by the release of the intracellular enzyme lactate dehydrogenase (LDH) into the cell culture supernatant 24 h after test compound addition using the Cytotoxicity Detection Kit (Roche Diagnostics, Rotkreuz, Switzerland).
7
Cell Viability Assay Protocols
8
Prostate Cancer Cell Line and PDX Cultivation
9
ATP assay for rotenone cytotoxicity
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PHH was plated at 1 × 105 in 96-well collagen-I-coated plates. At 24 and 168 h, serial concentrations (0–20 mM) of rotenone (Sigma-Aldrich, St. Louis, MO) were made in DMSO (Fisher Scientific). Then, the compound solutions were added to culture media at 1/200 (v/v) ratio to make final dosing concentrations of 0–100 µM (0.5 % v/v DMSO). Culture media were removed and replaced with the media containing rotenone. Following incubation for 2 h (37 °C, 5 % CO2), ATP content was assessed using the CellTitre-Glo assay (Promega, Madison, WI), according to the manufacturers’ instructions. Briefly, 30 µl of ATP reagent was added to each well containing 100 µl of media. The plate was shaken (1 min), and then, 100 µl of the well content was transferred to a white 96-well plate, and the ATPase luminescence was measured using a plate reader (Varioskan, Thermo Scientific, Waltham, MA). Results were presented as percentage of control.
10
Identifying Oncogenic Targets via siRNA
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Cells were transfected with four independent small interfering RNAs (siRNAs) targeting all genes in the 11p13 amplicon using Hiperfect (Qiagen, Crawley, UK). The sequences of the siRNA are detailed in the Supplementary Methods (available online). Cellular proliferation after siRNA treatment was assessed after 72 hours using the CellTitreGlo assay (Promega, Madison, WI, USA) and a plate luminescence reader (Tecan; Perkin-Elmer, Waltham, UK). Cell inhibition studies were performed as previously described (17 (link)) using everolimus (Sigma-Aldrich, Poole, UK), PP242 (Stratech Scientific Limited, Suffolk, UK), and wortmannin (Calbiochem, Nottingham, UK).
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