BAX−/−/BAK−/− MEF reconstituted with human wild type BAX were plated overnight (5,000 cells/well) in a 384-well opaque plate. After 18 hrs, the media was removed and 50 µL of fresh media was added. Cell were treated with BAI1 (5 µM, 2.5 µM, 1.25 µM) or DMSO for 2 hrs; compounds were dispensed using a TECAN D300 digital dispenser. After 2 hrs, media was removed and cells were washed 3 times with 1 XPBS. Following last wash, 1X PBS was removed from every well and 50 µL of staining solution was added directly into the plate. The staining solution contains a final concentration of 0.02 mg/mL oligomycin, 0.05 mg/mL digitonin, 2 µM JC-1, 0.01 M 2-mercaptoethanol dissolved in MEB solution. The MEB solution contains: 150 mM mannitol, 10 mM HEPES-KOH, 50 mM KCl, 0.02 mM EGTA, 0.02 mM EDTA, 0.1% BSA, 5 mM succinate, pH 7.5. After addition of staining solution, BIM BH3 peptide, CCCP, alamethicin and DMSO were added using TECAN D300 digital dispenser. Fluorescence was measured at 545 nm excitation and 590 nm emission using the M1000 microplate reader (TECAN) at 30°. Percentage of depolarization was calculated by normalization to the solvent-only control (0% depolarization) and the positive control CCCP (100% depolarization). Mitochondrial polarization analysis was performed in triplicates.
D300 digital dispenser
Manufactured by Tecan
Sourced in Switzerland
The D300 digital dispenser is a precision liquid handling instrument designed for small-volume dispensing. It offers accurate and reproducible liquid delivery in the nanoliter to microliter range, making it suitable for a variety of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
M1000 microplate reader, by Tecan (2 mentions)
Cytarabine, by Selleck Chemicals (1 mentions)
Daunorubicin, by Selleck Chemicals (1 mentions)
Etoposide, by Selleck Chemicals (1 mentions)
Navitoclax, by Selleck Chemicals (1 mentions)
Venetoclax, by Selleck Chemicals (1 mentions)
Elisa max deluxe set, by BioLegend (1 mentions)
Celltiter glo luminescent assay, by Promega (1 mentions)
6 protocols using d300 digital dispenser
1
Mitochondrial Polarization Assay for BAX/BAK KO MEFs
2
Mitochondrial Polarization Assay for BAX/BAK KO MEFs
3
Quantitative Viability Assay for Drug Screening
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Pyrvinium pamoate was purchased from MedChemExpress. Venetoclax, navitoclax, etoposide, cytarabine and daunorubicin were purchased from Selleckchem. All compounds were dissolved in DMSO and subsequently diluted in medium when used for viability assays.
For the validation of the most interesting hits from our drug screen and for drug combination studies, expanded dose response curves were made using the Tecan D300 Digital Dispenser (Tecan, Switzerland) to dispense the drug. The drug response on the cell viability was assessed by a 4-day MTT assay as described elsewhere [13] (link). Briefly, after 4 days of incubation, 5μL/well of 5 mg/ml MTT was added to 40μL of drug-exposed cells for 6 h. Thereafter, the reaction was stopped by adding 40μL/well of a 10% SDS/0.01 M HCL solution. The next day, the absorbance of the cells was measured at wavelengths 570 nm and 720 nm using the microplate reader (Versamax). The data of 720 nm served as background noise and was subtracted from the 570 nm data. MTT data was normalized to DMSO control, tolerating a maximum concentration of ≤0.5% (v/v). The value of optical density for the controls were at least 0.07 to ensure high viability of the primary cells when assessing the effect of the drug.
For the validation of the most interesting hits from our drug screen and for drug combination studies, expanded dose response curves were made using the Tecan D300 Digital Dispenser (Tecan, Switzerland) to dispense the drug. The drug response on the cell viability was assessed by a 4-day MTT assay as described elsewhere [13] (link). Briefly, after 4 days of incubation, 5μL/well of 5 mg/ml MTT was added to 40μL of drug-exposed cells for 6 h. Thereafter, the reaction was stopped by adding 40μL/well of a 10% SDS/0.01 M HCL solution. The next day, the absorbance of the cells was measured at wavelengths 570 nm and 720 nm using the microplate reader (Versamax). The data of 720 nm served as background noise and was subtracted from the 570 nm data. MTT data was normalized to DMSO control, tolerating a maximum concentration of ≤0.5% (v/v). The value of optical density for the controls were at least 0.07 to ensure high viability of the primary cells when assessing the effect of the drug.
4
T Cell Cytokine Profiling in Leukemia
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T cells/mL were stimulated with leukemic blasts at a ratio of 1:1. After 24 hours, culture supernatants were harvested and levels of INF-γ, TNF-α, and IL-2 were investigated with an enzyme-linked immunosorbent assay (ELISA MAX Deluxe Set, Biolegend, San Diego, CA, USA) according to the manufacturer’s instruction. The samples were read with an absorbance plate reader (Tecan, D300 Digital Dispenser, Männedorf, ZH, Switzerland). The limit of detection was 7.8 pg/mL.
5
Cytotoxicity Assay with MLN4924
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HEK293T–Cas9 cells were resuspended at 0.15 × 106 ml−1 treated with DMSO or 100 nM MLN4924 and plated on 384-well plates. After 2 h, the indicated drugs were dispensed with a D300 digital dispenser (Tecan). After 3 days of drug exposure, cell viability was assessed using the CellTiter-Glo luminescent assay (Promega, G7572) on an CLARIOstar Plus, MARS 3.4 (BMG LabTech). Cell viabilities were calculated relative to DMSO controls. The half-maximum effective concentration (EC50) values were derived from standard four-parameter log-logistic curves fitted with the ‘dr4pl’ R package.
6
Validating Drug Synergy Screening Protocol
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For the validation of the top hits from the high‐throughput drug screen and synergy studies, expanded dose response curves were made using the Tecan D300 Digital Dispenser (Tecan, Switzerland) to dispense the drug. Again the drug response on the cell viability was assessed by a MTT assay. MTT data were normalized to DMSO control, tolerating a maximum concentration of ≤0.5% (v/v). Experiments performed in triplicate for ALLPO and SEM, in duplo for KOPN8, with three technical replicates each.
Drug synergy between decitabine and the combined compounds was determined using BLISS independence model calculations [20 (link)], with the equation:E c o m b i = E A + E B − E A ∗ E B
Drug synergy between decitabine and the combined compounds was determined using BLISS independence model calculations [20 (link)], with the equation:
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