Cells were incubated with 100 μM 5-bromo 2′-deoxyuridine (BrdU) obtained from Sigma-Aldrich for 6 h as described [11 (link)]. The anti-BrdU antibody (Sigma B-2531) was obtained from Sigma-Aldrich and prepared at a 1:500 dilution. Additionally, to assess growth, 7500 cells per well were plated in triplicate in a 96-well plate. Cell mass was measured at 0, 3, and 5 days using colorimetric CellTiter 96 MTS assay (Promega). Experiments were repeated in three biologic replicates. Error bars represent standard deviation. Statistical significance was determined by Student’s t-test or by ANOVA, as analyzed in GraphPad Prism.
Celltiter 96 mts assay
Manufactured by Promega
Sourced in United States
The CellTiter 96 MTS assay is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. It utilizes a tetrazolium compound that is bioreduced by cells into a colored formazan product, which can be quantified by measuring the absorbance.
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Lab products found in correlation
5 bromo 2 deoxyuridine brdu, by Merck Group (2 mentions)
Celltiter glo assay, by Promega (2 mentions)
Bortezomib, by Promega (2 mentions)
Biotek elisa plate reader, by Agilent Technologies (1 mentions)
Incucyte live cell analysis system, by Sartorius (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Etoposide, by Merck Group (1 mentions)
Geneprint 10 system, by Promega (1 mentions)
S63845, by Apexbio (1 mentions)
Lenticrisprv2 system, by Addgene (1 mentions)
Umi 77, by Selleck Chemicals (1 mentions)
A1210477, by Apexbio (1 mentions)
Q vd oph, by Apexbio (1 mentions)
B2531, by Merck Group (1 mentions)
Nalm6, by American Type Culture Collection (1 mentions)
Easysep human t cell isolation kit, by STEMCELL (1 mentions)
Human il 2, by Thermo Fisher Scientific (1 mentions)
Jurkat, by Leibniz Institute DSMZ (1 mentions)
Kopn 8, by American Type Culture Collection (1 mentions)
20 protocols using celltiter 96 mts assay
1
Quantifying Cell Proliferation with BrdU
2
Cell Viability Assay after Transfection
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Cell viability after transfection was measured using CellTiter 96 MTS assay (Promega, Fitchburg, WI, USA) at 24, 48, and 72 hr after transfection. Briefly, the cells were incubated with MTS solution for half an hour, subsequently the absorbance at 490 nm was read from each well by using Biotek ELISA plate Reader (BioTek Instruments, Winooski, VT, USA). The assays were performed three times in triplicates for each group.
3
Trametinib-Induced Cell Proliferation Assay
4
Cell Viability Assay for MCL-1 Inhibitors
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Human cell lines were originally sourced from the American Type Culture Collection (ATCC) and were authenticated by Promega GenePrint 10 System (Promega WI, USA). Cells were maintained at 37 °C with 5% CO2 with 10% fetal bovine serum (FBS), except for in vitro experiments using A1210477 where FBS level was reduced to 3% during drug treatment and also in the relevant control samples. Cell viability was determined by CellTiter 96 MTS assay (Promega) after 48 h incubation with the indicated concentration of MCL-1 inhibitor UMI-77 (Selleck, UK), S63845 (Apexbio, UK) or A1210477 (Apexbio, UK). SYTOX Green (Invitrogen, UK) was used to identify dead cells and cell confluence measured using the Incucyte Live Cell Analysis System (Essen Bioscience, UK). 10 μM etoposide (Sigma, UK) was used to induce apoptosis and 10 μM Q-VD-OPh (Apexbio, UK) was used to block caspase activity. CRISPR/Cas9 gene editing using the LentiCRISPRv2 system (Addgene, MA, USA) was performed for BAX and BAK as described previously2 (link).
5
Cell Proliferation and Cytotoxicity Assay
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The CellTiter 96 MTS assay (Promega) was used to determine the cytotoxicity of the relevant drugs and cell proliferation according to the manufacturer’s instructions. For cell proliferation detection, cells were plated at the appropriate density (1 × 104 cells per well of a 96-well plate), and the proliferation of the indicated cells for 5 days was detected.
6
Cytotoxicity Assay for Bortezomib and Carfilzomib
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The CellTiter 96 MTS assay (Promega) was used to determine the cytotoxicity of the relevant drugs and cell proliferation, according to the manufacturer’s instructions. Cell viability was measured with MTS assay 24 hours after the addition of bortezomib or carfilzomib with graded concentrations in triplicate.
7
Evaluation of Proteasome Inhibitors in Myeloma Cells
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The CellTiter 96® MTS-assay (Promega) was used to determine the cell viability of bortezomib, (BTZ, obtained from Janssen Cilag), carfilzomib and CuEt in cell lines, according to manufacturer’s instructions, and the absorbance of the formazan product was measured in 96 well microplates at 492nm. The assay measures dehydrogenase enzyme activity found in metabolically active cells.
For patient cells, the more sensitive luminescent CellTiter-Glo assay (Promega) was used to determine cell viability, measured by ATP production of metabolically active cells. The primary myeloma cell samples were obtained after written informed consent and approval by the independent ethics review board (St. Gallen ethics committee - Ethikkommission Ostschweiz), in accordance with ICH-GCP and local regulations. Malignant plasma cells were retrieved by PBMC isolation from a patient with multiple myeloma progressing under BTZ containing therapy, based on IMWG criteria (“bortezomib resistant”) and an untreated patient (“bortezomib sensitive”). The purity of the cell samples was >80 % myeloma cells, as assessed by morphology.
For patient cells, the more sensitive luminescent CellTiter-Glo assay (Promega) was used to determine cell viability, measured by ATP production of metabolically active cells. The primary myeloma cell samples were obtained after written informed consent and approval by the independent ethics review board (St. Gallen ethics committee - Ethikkommission Ostschweiz), in accordance with ICH-GCP and local regulations. Malignant plasma cells were retrieved by PBMC isolation from a patient with multiple myeloma progressing under BTZ containing therapy, based on IMWG criteria (“bortezomib resistant”) and an untreated patient (“bortezomib sensitive”). The purity of the cell samples was >80 % myeloma cells, as assessed by morphology.
8
NSCLC Cell Migration Assay
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Measurement of in vitro NSCLC cell migration was performed, as we have previously described (30 (link)). Twenty-four transwell units with 8 μm pore size (Millipore, Billerica, MA, USA) were used for monitoring in vitro cell migration. Control or HABP2 overexpressing cells (5 × 103 cells/well) were plated in the upper chamber and incubated with 0.2 ml of serum-free media containing either vehicle (control), 100 nM LMW-HA or 100 nM HMW-HA with or without 1 h pretreatment with 1 μM of the uPA inhibitor UK122 and media with serum was added to the lower chamber. Cells were allowed to migrate through the pores for 18 h. Cells from the upper and lower chamber were quantitated using the CellTiter96™ MTS assay (Promega, San Luis Obispo, CA, USA) and read at 492 nm. Percent migration was defined as the number of cells in the lower chamber divided by the number of cells in both the upper and lower chamber. Each assay was set up in triplicate and repeated at least five times.
9
Colorimetric Cell Viability Assay
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Cell viability was measured using the colorimetric CellTiter 96® MTS assay (Promega, G1111) that we have adapted for 24-well format. Phenazine methosulfate (PMS, P9625) was made up in DPBS (0.92 mg/mL), sterile filtered and stored at −20 °C before use. Briefly, after removing culture supernatants, cells were washed once with PBS and 250 μL of freshly prepared phenol red-free medium containing MTS-PMS (20:1) was added. The cells were incubated for 0.5–1.5 h at 37 °C, after which 100 μL of the culture supernatant was transferred to a 96-well for measurement of absorbance at 490 nm in a microplate reader. Data was normalized using cells untreated with compounds (100%) and buffer only background controls (0%).
10
MTS Assay for Cytotoxicity Evaluation
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The CellTiter 96 MTS assay (Promega) was used to determine the cytotoxicity of the relevant drugs and cell proliferation, in accordance with the manufacturer's instructions. Cell viability was measured using the MTS assay 24 h after the addition of drug with graded concentrations in triplicates.
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