In vitro, the cell sensitivity to each antineoplastic drug was determined by a modified MTT assay [12 (link),35 ]. Briefly, each well of 96 well plates contained approximately 3000 H460 cells or H460/MX20 cells. Cells were collected, re-suspended in media, and seeded evenly in 96-well plates and incubated for 24 h at 37°C. Then, 20 μL of a predetermined, fixed concentration of cabozantinib was added into each well. After 1 h of incubation, 20 μL of the respective antineoplastic drugs were added into each well, serially diluted. After 72 h of incubation, 20 μL of 4mg/ml MTT solution was added to each well. The plates were incubated at 37°C for 4 hours in the presence of the MTT solution. Finally, the MTT-medium was removed and 100 μl DMSO was added to each well to terminate the staining. Absorbance values of the samples were measured at 570 nm using Opsys microplate reader (Dynex Technologies, Chantilly, VA).
Opsys microplate reader
Manufactured by Dynex
Sourced in United States
The Opsys microplate reader is a versatile instrument designed for the analysis of various biological and biochemical assays in a microplate format. It is capable of performing absorbance, fluorescence, and luminescence measurements across multiple wavelengths. The core function of the Opsys microplate reader is to provide precise and reliable data acquisition for a wide range of applications, enabling researchers to conduct efficient and accurate experiments.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Paclitaxel, by Merck Group (2 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Eclipse te 2000 s fluorescence microscope, by Nikon (1 mentions)
Papain from papaya latex, by Merck Group (1 mentions)
Blyscan assay, by Biocolor (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Cyclosporine a, by Merck Group (1 mentions)
Phosphate buffered saline 20 concentrate ph 7.5, by Avantor (1 mentions)
3 4 5 dimethylthiazol yl 2 5 diphenyltetrazoliumbromide mtt dimethyl sulfoxide dmso, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Alexa flour 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Vincristine, by Merck Group (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Verapamil, by Merck Group (1 mentions)
32 protocols using opsys microplate reader
1
MTT Assay for Drug Sensitivity
2
Quantifying Anticancer Drug Sensitivity
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An MTT colorimetric assay with minor modifications from that previously described11 was used to detect the sensitivity of cells to anticancer drugs. Cells were harvested after addition of trypsin and suspended at a concentration of 6×103 cells/well. For the reversal experiment, PD173074 (0.25 or 1 µmol/L, 20 µL/well) or cepharanthine (2.5 µmol/L, 20 µL/well) was added, followed by different concentrations of chemotherapeutic drugs (20 µL/well) into designated wells. After 68 h of incubation, 20 μL of MTT solution (4 mg/mL) was added to each well, and the plate was further incubated for another 4 h, allowing viable cells to convert the yellow-colored MTT into dark blue formazan crystals. Subsequently, the medium was aspirated, and 100 μL DMSO was added to each well to dissolve the formazan crystals. The absorbance was determined at 570 nm by an OPSYS Microplate Reader (DYNEX Technologies, Chantilly, VA). The degree of resistance was calculated by dividing the IC50 (concentrations required to inhibit growth by 50%) for the MDR cells by that of the parental sensitive cells. The IC50 values were calculated to construct the survival curves using the Bliss method12 .
3
Assessing Drug Sensitivity in Multidrug-Resistant Cell Lines
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To determine the drug sensitivities of the previously described ABCB1-overexpressing KB-C2 cells, LLC-MDR1-WT cells, and ABCC10-overexpressing HEK293/ABCC10 cells, with KB-3-1, LLC-PK1, and HEK293/pcDNA3.1 cells as the respective controls [1 (link),16 (link)], a modified MTT assay was performed [1 (link),21 (link)]. Approximately 4,000 KB-3-1 cells, 7,000 KB-C2 cells, and 5,000 LLC-PK1, LLC-MDR1-WT, HEK293/pcDNA3.1, and HEK293/ABCC10 cells were seeded in 180 μL of medium in each well of 96-well plates. After incubating for 24 h at 37°C, 20 μL of paclitaxel or cabazitaxel (0.01 to 10 μmol/L) was added. Subsequently, cells treated with paclitaxel or cabazitaxel in DMEM supplemented with 10% FBS were incubated at 37°C for 72 h. After 72 h, 20 μL MTT (4 mg/mL) was added to each well. The cells were incubated at 37°C for another 4 h. The MTT with medium was removed, and 100 μL of DMSO was added to each well. The absorbance was measured at 570 nm by an Opsys microplate reader (Dynex Technologies, VA, USA). The degree of resistance was calculated by dividing the 50% inhibition concentration (IC50) as calculated using the Bliss method for drug-resistant cells (KB-C2, LLC-MDR1-WT, and HEK293/ABCC10) by that of the parental drug-sensitive cells (KB-3-1, LLC-PK1, and HEK293/pcDNA3.1), respectively. Each MTT assay was run in triplicate.
4
CCK-8 Assay for Cancer Cell Proliferation
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The proliferation of cancer cells was tested by CCK-8 assay. Briefly, cells were seeded in 96-well plate with about 5 × 103 cells in each well. The cells were treated with tested compounds after 24 h of incubation. CCK-8 reagent (10 ml) was added to each well after 72 h of incubation, and cells were incubated at 37 °C for 4 h. The light absorbance at 450 nm was measured by using an Opsys microplate reader (Dynex Technologies, Chantilly, VA, USA). Results are illustrated as percent of cell viability normalised to DMSO-treated control cells.
5
Quantifying Anticancer Drug Sensitivity
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The MTT colorimetric assay was used to detect the sensitivity of cells to anticancer drugs. Cells were harvested with trypsin treatment. After washing with PBS, cells were resuspended in the culture media. Cells with a final concentration of 5 × 103 cells/well were seeded evenly into 96-well plates with 160 μL media. For the reversal experiments, SSJ compounds, verapamil, FTC or PAK104P (20 μL/well) was added followed by different concentrations of chemotherapeutic drugs (20 μL/well) into designated wells. After 72 h of incubation, 20 μL of MTT solution (5 mg/mL) was added to each well, and the plate was further incubated for 4 h, allowing viable cells to convert the yellow colored MTT into dark-blue formazan crystals. Subsequently, the medium was discarded, and 100 μL of dimethylsulfoxide (DMSO) was added into each well to dissolve the formazan crystals. The absorbance was determined at 570 nm by an OPSYS Microplate Reader from DYNEX Technologies Inc. (Chantilly, VA, USA) followed previously described protocol [22 (link)]. The fold of resistance was calculated by dividing the IC50 (concentrations required to inhibit growth by 50%) of the MDR cells by that of the parental sensitive cells.
6
MTT Colorimetric Assay for Drug Sensitivity
7
Evaluation of HEK293 Cells' Sensitivity to Anticancer Drugs
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The viability of HEK293/pcDNA3.1 and HEK293/ABCB1 cells to anticancer drugs was measured using the MTT assay. Cells (5000 cells/well) were seeded in 96-well plates (160 μL/well) and cultured for 24 h. For the cytotoxicity experiment of Y6, 40 μL of varying concentrations of Y6 were added into each well to the final concentrations of: 100,30,10,3,1,0.3,0.1,0.03 μmol/L. They were incubated for 48 h at 37 °C. For the reversal experiment, Y6, EGCG, and the control modulator (verapamil) (20 μL/well) were added 1 h prior. Then, twenty microliters of different concentrations of chemotherapeutic drugs (doxorubicin or cisplatin) were added into the designated wells and incubated for 48 h at 37 °C. Subsequently, 20 μL of the MTT solution (4 mg/mL) was added to each well and incubated for an additional 4 h. The solution was discarded and 100 μL of DMSO were added to dissolve the formazan crystals. Finally, light absorbance was determined at 490 nm using the OPSYS microplate reader (Dynex Technology, Chantilly, VA, USA). Verapamil (1 μmol/L) was used as positive control inhibitors of ABCB1.
8
MTT Colorimetric Assay for Anticancer Drug Sensitivity
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The modified MTT colorimetric assay was used to detect the sensitivity of cells to anticancer drugs in vitro [51 (link)]. Cells were seeded in 180 μl of medium in 96-well plates in triplicate at 5000–6000 cells/well. After incubation at 37°C for 24 h, the cells were treated with different concentrations of chemotherapeutic drugs (20 μl/well). After 72 h of incubation at 37°C, 20 μl of MTT solution (4 mg/ml) was added to each well. The plates were further incubated at 37°C for 4 h, allowing viable cells to change the yellow-colored MTT into dark-blue formazan crystals. Subsequently, the MTT/medium was removed from each well without disturbing the cells, and 100 μl of DMSO was added into each well. Plates were placed on a shaking table to thoroughly mix the formazan into the solvent. Finally, the absorbance was determined at 570 nm by Opsys microplate reader (Dynex Technologies, Chantilly, VA).
9
Investigating ABCB1, ABCG2, ABCC1, and ABCC10 Mediated Drug Resistance
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The MTT assay was carried out to determine the cytotoxicity of BMS-599626 in parental and ABCB1-, ABCG2-, ABCC1- and ABCC10-overexpressing sublines. Cells were trypsinized, resuspended and 5 × 103 cells per well were seeded in a 96-well plate. After 24 h, the cells were incubated with or without drugs for 72 h preceded by a 2 h treatment with either BMS-599626 or Ko143. MTT (3 mg/mL) was added to the cells and the cells were further incubated for another 4 h. Subsequently, the supernatant was removed and dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was read in an Opsys microplate reader (Dynex Technologies, Chantilly, VA) at an absorbance of 570 nm. The concentration at which 50% of cell growth was inhibited (IC50) was determined as illustrated previously [8 (link),18 (link)]. The ratio of the IC50 of the resistant cells to the IC50 of sensitive cells yielded the resistant folds (Rf). The concentrations of BMS-599626 used for the reversal studies were 100 and 300 nM. Ko143 was used as positive control for inhibition of ABCG2. Cytotoxicity assays were conducted in triplicates and each assay was run at least three times.
10
ABCG2 Reversal Study: MTT Assay
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The viability of NCI-H460 and NCI-H460/MX20 cells or HEK293/pcDNA3.1 and HEK293/ABCG2-482-R2 cells to chemotherapeutic drugs was measured for the ABCG2 reversal study using the MTT assay. Cells (5000 cells per well) were seeded into 96-well microplates with 160 μL per well and cultured overnight. Mitoxantrone, SN-38, topotecan, and cisplatin were diluted with PBS to a series of various concentrations and added to designated wells (20 μL per well) with or without the reversal agents (Y6 and FTC) (20 μL per well). Cells were incubated for 72 h at 37°C. Then, 20 μl of MTT solution (4 mg/mL, in PBS) was added into each well and incubated for 4 h at 37°C in dark. Then the supernatant medium was discarded and 100 μl of DMSO was added to each well for formazan dissolution. Finally, the light absorbance at 490 nm was measured using the OPSYS microplate reader (Dynex Technology, Chantilly, VA, United States). FTC was used as positive reversal agent of ABCG2.
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