To evaluate the toxicity, mouse liver and ovarian organoids were plated in 96 multi well plates and treated with AgNP, AgNO3 and SNP ranging from 100 μg/ml to 0.034 μg/ml for 96h. As a positive control, we used CisPt starting from 30 μg/ml (100 μM) to 0.009 μg/ml (0.03 μM). After 96 h, organoids viability was measured by CellTiter-Glo® 3D Luminescence assay (Promega, Madison, WI, USA) with a Tecan M1000 instrument (Tecan, Mannedorf, Switzerland).
Celltiter glo 3d luminescence assay
Manufactured by Promega
Sourced in United States
The CellTiter-Glo® 3D Luminescence assay is a cell viability assay designed to measure the ATP levels in 3D cell cultures. The assay generates a luminescent signal that is proportional to the amount of ATP present, which is an indicator of the number of metabolically active cells in the sample.
Automatically generated - may contain errors
Lab products found in correlation
M1000 instrument, by Tecan (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by BD (1 mentions)
Basic fibroblast growth factor (bfgf), by Miltenyi Biotec (1 mentions)
Mammocult human medium, by STEMCELL (1 mentions)
Heparin, by STEMCELL (1 mentions)
Hydrocortisone, by STEMCELL (1 mentions)
Infinite 200 pro instrument, by Tecan (1 mentions)
3 protocols using celltiter glo 3d luminescence assay
1
Organoid Toxicity Assays for AgNP, AgNO3 and SNP
2
Spheroid Formation and Viability Assay
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Spheroids were dissociated in 100 µl of MammoCult™ Human Medium (STEMCELL Technologies) and seeded into 96-well ultra-low attachment plates (Corning, 7007). Cell viability of each spheroid was measured after 10–14 days by using CellTiter-Glo 3D luminescence assay (Promega, G9683).
To measure the number of spheroids, dissociated cells were grown in 6-well ultra-low attachment plates (Corning, 3471). Sphere media was first prepared by serum-free DMEM-F12 (Gibco), supplemented with B27 (1:50, Invitrogen), 20 ng/ml EGF (BD Biosciences), 20 ng/ml bFGF (Miltenyi Biotec) and 4 µg/ml heparin (STEMCELL Technologies), 0.24 µg/ml hydrocortisone (STEMCELL Technologies, and 1% antibiotics. An equal number of cells were cultured to generate spheres, and the number of spheres (diameter > 50 mm) was counted on days 5–7 under a microscope.
To obtain enough cells from the spheroids for the above assays, dissociated cells were cultured at ultra-low dishes (Corning, 3262) for the same period. Spheroids were collected by gentle centrifugation (30×g) and dissociated into single cells using 0.05% Trypsin. Cells were then filtered through 70-µm strainers and plated for the next generation of spheres.
To measure the number of spheroids, dissociated cells were grown in 6-well ultra-low attachment plates (Corning, 3471). Sphere media was first prepared by serum-free DMEM-F12 (Gibco), supplemented with B27 (1:50, Invitrogen), 20 ng/ml EGF (BD Biosciences), 20 ng/ml bFGF (Miltenyi Biotec) and 4 µg/ml heparin (STEMCELL Technologies), 0.24 µg/ml hydrocortisone (STEMCELL Technologies, and 1% antibiotics. An equal number of cells were cultured to generate spheres, and the number of spheres (diameter > 50 mm) was counted on days 5–7 under a microscope.
To obtain enough cells from the spheroids for the above assays, dissociated cells were cultured at ultra-low dishes (Corning, 3262) for the same period. Spheroids were collected by gentle centrifugation (30×g) and dissociated into single cells using 0.05% Trypsin. Cells were then filtered through 70-µm strainers and plated for the next generation of spheres.
3
PDTO Toxicity Assay of AgNP, AgNO3, and SNP
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Toxicity assay on PDTO was conducted at passage zero. PDTO were plated on 96 multi wells and treated with AgNP, AgNO3, and SNP ranging from 100 μg to 0.034 μg per ml for 96h. CisPt ranging from 30 μg/ml (100 μM) to 0.009 μg/ml (0.03 μM) for 96h. Organoid viability was evaluated by CellTiter-Glo® 3D Luminescence assay (Promega, Madison, WI, USA) with an Infinite200 PRO instrument (Tecan, Mannedorf, Switzerland). IC50 values were analysed from a non-linear regression method using GraphPad prism software.
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