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3d cell titer glo

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The 3D Cell Titer Glo is a luminescent cell viability assay that quantifies the ATP present in metabolically active cells. It provides a quantitative measure of cell proliferation and cytotoxicity in three-dimensional cell culture models.

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Lab products found in correlation

Glomax 96 microplate luminometer, by Promega (4 mentions) Caspase glo 3 7, by Promega (3 mentions) White opaque 96 well plate, by Corning (3 mentions) Mx m 96 well plate shaker, by Scilogex (3 mentions) Celltiter glo, by Promega (2 mentions) Matrigel, by Corning (2 mentions) Tryple, by Thermo Fisher Scientific (2 mentions) Fulvestrant, by Selleck Chemicals (2 mentions) Prism 9, by GraphPad (2 mentions) Ultra low attachment 96 well plate, by Corning (1 mentions) Fluoroskan ascent fl reader, by Thermo Fisher Scientific (1 mentions) Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions) Cytation 3 imaging reader, by Agilent Technologies (1 mentions) Cellstar, by Greiner (1 mentions) Rock inhibitor, by Selleck Chemicals (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Prism 7, by GraphPad (1 mentions) Luminoskan ascent, by Thermo Fisher Scientific (1 mentions) Ab16667, by Abcam (1 mentions) Cck 8 assay, by Dojindo Laboratories (1 mentions)

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Cell Titer Glo 3D Assay kit (3 citations)

20 protocols using 3d cell titer glo

1

Evaluating TNBC Tumorsphere Viability

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The TNBC cells were cultured in NanoCulture plates (2 × 104/well, SCIVAX Corporation, Kanagawa, Japan) and 96-well ultralow attachment plates (4 × 103/well, Costar®, New York, NY, USA). Cells transfected with 10 nM scramble or siRNA against DYRK1B were cultured for 7 d to observe tumorsphere of the control and silenced tumorsphere under microscope. Tumorsphere viability was evaluated by 3D CellTiter Glo (G9681, Promega) or Calcein AM (Green)/Ethidium homodimer-1 (EthD-1, Red) (LIVE/DEAD® Viability/Cytotoxicity Kit, ThermoFisher Scientific) to differentiate live and dead cells. Tumorsphere viability was evaluated and quantitated by Fluoroskan Ascent FL reader (Thermo Fisher Scientific), as previously described [17 (link)].
Chang C.C., Chiu C.C., Liu P.F., Wu C.H., Tseng Y.C., Lee C.H, & Shu C.W. (2021). Kinome-Wide siRNA Screening Identifies DYRK1B as a Potential Therapeutic Target for Triple-Negative Breast Cancer Cells. Cancers, 13(22), 5779.
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2

Metabolic Activity Assays in 2D, 3D, and Co-cultures

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Metabolic activity assays were performed on HCT116, DLD1 and SW620 cells grown in 2D, 3D and 3D co-cultures. Post-treatment cell viability was measured using the luminescent-based cell metabolic activity assay CellTiter-Glo® (G7572, Promega) for 2D cultures and 3D-CellTiter-Glo® (G9683, Promega) for 3D and 3D co-cultures, according to the manufacturer’s instructions. The intensity of the luminescence signal was detected with the BioTek Cytation 3 imaging reader with corresponding Gen5 Image software version 3.04, using standard settings.
Zoetemelk M., Rausch M., Colin D.J., Dormond O, & Nowak-Sliwinska P. (2019). Short-term 3D culture systems of various complexity for treatment optimization of colorectal carcinoma. Scientific Reports, 9, 7103.
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3

Cytotoxicity and Apoptosis Assays

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Measurement of the toxicity profile was performed using viability (ATP measurement) and apoptosis (3/7 caspase activity) endpoints kits 3D Celltiter-Glo and Caspase-Glo 3/7 (Promega), respectively. After each time point of exposure, medium was removed from the plates and replaced by 100 µL of each kit reagent to the appropriate wells, followed by homogenization of the Matrigel. The plates were placed in a Scilogex MX-M 96 well plate shaker for 1 h (incubation time), at room temperature. Afterwards, samples were transferred to white opaque 96-well plates (Corning) and luminescence was measured in GloMax® 96 Microplate Luminometer (Promega). Luminescence values corresponding to the levels of either ATP or 3/7 caspase activation, were transferred to Excel and corrected for the blank reaction to eliminate possible interferences of the Matrigel matrix in both curves.
Rodrigues D., de Souza T., Coyle L., Di Piazza M., Herpers B., Ferreira S., Zhang M., Vappiani J., Sévin D.C., Gabor A., Lynch A., Chung S.W., Saez-Rodriguez J., Jennen D.G., Kleinjans J.C, & de Kok T.M. (2021). New insights into the mechanisms underlying 5-fluorouracil-induced intestinal toxicity based on transcriptomic and metabolomic responses in human intestinal organoids. Archives of Toxicology, 95(8), 2691-2718.
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4

Matrigel-Based 3D Cell Culture Assay for Sulfopin and Sulfopin-AcA

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PATU-8988T (WT or Pin1−/−) Matrigel suspensions were prepared by resuspension of cells in 100% cold Matrigel (kept on ice). One dome was plated per well in a 24-well plate (Greiner CELLSTAR), with 50 μl of cells per Matrigel suspension per dome and 1,000 cells per dome. After plating the domes, the 24-well plate was placed on top of a T175 (previously filled with autoclaved water) in an incubator at 37 °C and 5% CO2, and left for 15 min for the domes to solidify. Keeping the 24-well plate on the T175 filled with water, the plate was then transferred to a tissue culture hood and 500 μl of cold DMEM (+10% FBS/1% penicillin/streptomycin) was added to each well. The plate was then returned to the incubator. The next day, either DMSO, Sulfopin (1 μM) or Sulfopin-AcA (1 μM) was added to the corresponding wells. Every 3 days the medium was carefully aspirated off (so as not to disrupt the Matrigel domes) and 500 μl of fresh medium/compound was added. After 9 days the medium was aspirated off and 300 μl of 3D CellTiter-Glo (Promega, catalog no. G9681) was added to each well. After shaking the plate for 1 h at room temperature, luminescence was measured using an Envision plate reader; n = 9 biological replicates were used for each treatment condition.
Dubiella C., Pinch B.J., Koikawa K., Zaidman D., Poon E., Manz T.D., Nabet B., He S., Resnick E., Rogel A., Langer E.M., Daniel C.J., Seo H.S., Chen Y., Adelmant G., Sharifzadeh S., Ficarro S.B., Jamin Y., da Costa B.M., Zimmerman M.W., Lian X., Kibe S., Kozono S., Doctor Z.M., Browne C.M., Yang A., Stoler-Barak L., Shah R.B., Vangos N.E., Geffken E.A., Oren R., Koide E., Sidi S., Shulman Z., Wang C., Marto J.A., Dhe-Paganon S., Look T., Zhou X.Z., Lu K.P., Sears R.C., Chesler L., Gray N.S, & London N. (2021). Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo. Nature chemical biology, 17(9), 954-963.
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5

3D Cell Culture for Sphere Formation

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Three-dimensional cell culture was performed as reported previously with minor modifications [56 (link)]. NanoCulture system was used for 3D cell culture (organogenix). First, 100 ul of medium was added to each well. The plate was then centrifuged at 2000 g for 5 min to remove microbubbles, followed by incubation for 15–30 min at incubator. Each well was seeded with 50 ul of medium containing 5 × 103 cells (If needed, transfection was conducted at this time point.). The plate was then kept on the bench at room temperature for 10–15 min until cells adhered to the bottom film. At 24 h, 50 μl of medium (including drug) was added for 4–6 days until observation of sphere formation. Pictures of the spheres were taken under microscope and cell viability was measured with 3D Cell Titer Glo (Promega, Madison, WI, USA).
Tsai W.L., Cheng J.S., Liu P.F., Chang T.H., Sun W.C., Chen W.C, & Shu C.W. (2022). Sofosbuvir induces gene expression for promoting cell proliferation and migration of hepatocellular carcinoma cells. Aging (Albany NY), 14(14), 5710-5726.
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6

ER+ Patient-Derived Xenograft Organoid Drug Screening

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ER+ PDX organoids were established as previously described69 (link). For drug testing studies, organoids were dissociated with Tryple (Gibco, MA, USA) at 37 °C for 30 mins in the presence of 10 µM Rock inhibitor (Selleckchem, TX, USA). After counting the single cells, they were plated into 96-well plate (20,000 cells/well) on a matrigel-coated surface with media containing 2% matrigel (Corning, NY, USA). SOC therapies with or without PDE4D or EGFR or PARP1 inhibitors were added 72 hours after seeding. Organoids were grown in the presence of drugs or vehicle for 7 days, and the organoid viability was measured using 3D Cell Titer Glo (Promega, WI, USA).
Saatci O., Cetin M., Uner M., Tokat U.M., Chatzistamou I., Ersan P.G., Montaudon E., Akyol A., Aksoy S., Uner A., Marangoni E., Sajish M, & Sahin O. (2023). Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer. Nature Communications, 14, 6997.
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7

Estrogen-Responsive Organoid Viability Assay

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For both estrogen responsiveness and dependence, organoids were digested and plated as above 40–50,000 cells/ml, depending on the organoid line. Three days after plating, media was refreshed, and wells were treated with either 1.0 nM of b-estradiol or vehicle control (EtOH) using the Tecan D300e drug dispenser. For dependence, media was replaced with fresh BTOM-ER or phenol red-free BTOM-ER. Media was refreshed every 2–3 days. After 10 days, organoids were assessed for viability using 3D Cell Titer Glo (Promega). Viability measurements were analyzed using GraphPad Prism software.
Oliphant M.U., Akshinthala D, & Muthuswamy S.K. (2024). Establishing conditions for the generation and maintenance of estrogen receptor-positive organoid models of breast cancer. Breast Cancer Research : BCR, 26, 56.
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8

Doxycycline-Induced 2D and 3D Cell Viability

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1,000 MIA PaCa-2 cells were seeded in white 96 well plates (clear bottom, Revvity, Cat#6005181) for 2D proliferation assays or in ultra-low attachment 96 well plates (Corning, Cat#7007) for 3D spheroid assays. The cell seeding was optimized to maintain linear growth over the time of the assay. The following day doxycycline was prepared at 10X concentration (5 μg mL−1 for 0.5 μg mL−1 final concentration). Cells were incubated in the presence of the doxycycline for 6 days. Cell viability was determined every two days using CellTiter-Glo (Promega, Cat#G7572) or 3D CellTiter-Glo (Promega, Cat#G9683) by incubation with the cells for 15 min. Cell viability was determined by normalizing doxycycline treated cells to non-treated cells at day 6. Cells from the ultra-low attachment plates were transferred into a white 96 well plate (Greiner, Cat#655075). Plates were read on a CLARIOstar instrument.
Cornebois A., Sorbara M., Cristol M., Vigne E., Cordelier P., Desrumeaux K, & Bery N. (2024). Discovery of SOCS7 as a versatile E3 ligase for protein-based degraders. iScience, 27(5), 109802.
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9

Fulvestrant Treatment of Organoid Cultures

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Organoids were prepared as above, and on day three, media was refreshed, and wells were treated with either vehicle control or indicated concentrations of fulvestrant (Selleckchem) using the Tecan D300e drug dispenser. Media was refreshed, and plates were retreated every 2–3 days. After 5 days of treatment, organoids were assessed for viability using 3D Cell Titer Glo (Promega).
Oliphant M.U., Akshinthala D, & Muthuswamy S.K. (2024). Establishing conditions for the generation and maintenance of estrogen receptor-positive organoid models of breast cancer. Breast Cancer Research : BCR, 26, 56.
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10

Cytotoxicity Evaluation of CHD1Li

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The CHD1Li were tested at seven different concentrations to determine their cytotoxic effect on tumor organoids as previously described [24 (link),27 (link)]. Compounds were dissolved in DMSO (Sigma-Aldrich, Ref #34869) to make a 10 mM stock solution and stored at − 80°C. After thawing, the stock was diluted with growth media and serially diluted before addition to cells in a 1:6 ratio. After 72 h of growth, cells were treated with CHD1Li in triplicates or quadruplicates at final concentrations ranging from 0.625 μM to 40 μM in 0.4% DMSO. The plate was incubated at 37 °C for 72 h before harvesting. During harvesting, the organoids were transferred to a white, flat bottom 96-well plate (Greiner, Ref#:655083) and treated with 35 μL of 3D Cell Titer Glo (Promega, Ref#: G7572). The plates were then placed on an orbital shaker at 400 RPM for 45 min before they were analyzed using the Envision plate reader. These experiments were conducted on 2 to 3 separate instances on different days to determine result repeatability.
Clune S., Awolade P., Zhou Q., Esquer H., Matter B., Kearns J.T., Kellett T., Akintayo D.C., Kompella U.B, & LaBarbera D.V. (2023). The validation of new CHD1L inhibitors as a therapeutic strategy for cancer. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 170, 116037.
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