CellTiter-Glo Luminescent Cell Viability assays (Promega, Madison, WI) were performed on patient samples and cell lines grown in the conditions described above. Cell lines were seeded in 96-well plates at a concentration of 0.4 million cells/mL and treated with iCRT14 (0-40 μM). A panel of chemotherapeutic agents used in the therapy of ALL was added concurrently at hour 0 for the concurrent assays (etoposide, prednisolone and doxorubicin) and after 48 hours of iCRT pretreatment for the pretreatment assays (etoposide, prednisolone, doxorubicin, cytarabine and 6TG). iCRT14 doses were added daily to the cells in the pretreatment assays. CellTiter-Glo reagent was added and luminescence was recorded using a Flexstation 3 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) and the SoftMax Pro Data Acquisition and Analysis Software (Molecular Devices, Sunnyvale, CA, USA) at hour 24 for the concurrent assays and hour 72 for the pretreatment assays. Drug combination effects were analyzed by the Calcusyn software (Biosoft, Cambridge, GB, UK) according to the combination index (CI) equation of Chou-Talalay (Chou 2010 (link)). This analysis describes the drug interaction as: CI >1.1 antagonistic, 0.9-1.1 additive, <0.9 synergistic. Patient samples were pretreated with iCRT14 for only 24 hours because of their known decreased viability.
Softmax pro data acquisition and analysis software
Manufactured by Molecular Devices
Sourced in United States
SoftMax Pro Data Acquisition and Analysis Software is a comprehensive data management tool designed for use with Molecular Devices' microplate readers. The software enables users to acquire, analyze, and report data generated from a variety of experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Flexstation 3 microplate reader, by Molecular Devices (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Calcusyn software, by Biosoft (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase system, by Promega (1 mentions)
Spectramax id5, by Molecular Devices (1 mentions)
Flipr calcium 6 assay kit, by Molecular Devices (1 mentions)
Streptavidin alkaline phosphatase, by Southern Biotech (1 mentions)
Spectramax plus 384, by Molecular Devices (1 mentions)
Softmax pro, by Molecular Devices (1 mentions)
7 protocols using softmax pro data acquisition and analysis software
1
Assessing Synergy of iCRT14 and Chemotherapies
2
Wnt Pathway Activation and Inhibition Assay
3
Calcium Imaging Assay in 96-well Plates
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Cells were grown in phenol red free DMEM/F-12 media with 15 mM HEPES and L-glutamine with 10% FBS and 400 μg/mL G418 (or antibiotic-antimycotic, 1X, for HEK293 control cells). Cells were re-plated into clear bottom black tissue culture-treated 96-well plates (Thermo Scientific) at a density to achieve a confluency of 90% or greater at time of assay. Cells were treated with FLIPR Calcium 6 Assay Kit (Molecular Devices) following manufacturer instructions. Per kit protocol, reagents were equilibrated to room temperature and loading buffer was made by fully dissolving component A into component B. Equal volume of loading buffer was then added directly to culture media (100 μL of buffer to 100 μL of culture media in each well). Plate was incubated at 37°C for 2 h. Measurement was performed on SpectraMax iD5 using SoftMax Pro Data Acquisition and Analysis Software (Molecular Devices).
4
Measurement of Mouse Antibody Levels
5
ELISA for Thyroid Hormones
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Stop solutions for TSH, T4, and T3 ELISA were added, and the absorbance was measured at 450 nm (SoftMax Pro Data Acquisition and Analysis Software, RRID: SCR_014240; Molecular Devices, San Jose, CA, USA). All measurements were performed in duplicates.
6
Adiponectin Receptor Quantification by ELISA
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Mouse AdipoR1 specific antibody-coated, 96-well enzyme-linked immunosorbent assay (ELISA) plates and AdipoR2 specific antibody-coated, 96-well ELISA plates (Sanghai Sunred Biological Technology Co., Ltd., Shanghai, PRC) were used following the manufacturer's instructions. For the control and four stress-exposed groups of both mouse strains in our experiment, three protein samples were randomly selected from the six available samples. The total protein concentration loaded into each well was 20 μg/ml in a 40 μl volume. The final OD values were measured with the SpectraMax® Plus384 (Molecular Devices, LLC., Sunnyvale, CA, USA) absorbance microplate reader and sample protein concentration was calculated with the SoftMax® Pro Data Acquisition and Analysis Software (Molecular Devices, LLC., Sunnyvale, CA, USA).
7
FcγR-binding Activity of Polatuzumab Vedotin and Surrogates
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The Fcγ receptor‐binding activities of polatuzumab vedotin, the unconjugated clinical antibody (the anti‐human CD79b mAb used for polatuzumab vedotin conjugation), the surrogate ADC, and the unconjugated surrogate antibody (the anti‐cynomolgus monkey CD79b mAb used for surrogate ADC conjugation) were measured by elisa using a panel of recombinant human Fcγ receptors (IA, IIA‐H131, IIA‐R131, IIB, IIIA‐F158, and IIIA‐V158) as previously described (Chung et al., 2012 ). For measuring binding to human FcγRII and FcγRIII, test antibodies were preincubated with anti‐human κ light chain F(ab′)2 to form a complex to increase binding avidity. Briefly, test antibodies were serially diluted and then added to plates coated with recombinant human FcγRs. Bound antibodies were detected using goat F(ab′)2–anti‐human, F(ab′)2–HRP, and tetramethylbenzidine (TMB). Dose–response binding curves were generated by plotting the mean absorbance values from duplicates of sample dilutions against the sample concentrations and fitted with a four‐parameter model using SoftMax Pro (Molecular Devices [SoftMax Pro Data Acquisition and Analysis Software, RRID:SCR_014240]). For comparison, the EC50 value of the reference molecule was set at 1, and the relative activity of each sample was calculated as follows:
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