Materials and Methods
The Epoch™ Multi-Volume Spectrophotometer System from BioTek Instruments was used for all absorbance measurements. The Take3™ plate accessory, which is included in the system, was used for all microvolume and 1 cm pathlength absorbance measurements.
The Take3 plate has a standard SBS footprint and 16 microspots arranged as columns akin to those of a 96-well microplate. Samples of 2–5 µL may be pipetted into individual microspots, or a multichannel pipette may be used to load eight samples or blanks simultaneously, allowing both to be run at the same time.
The Take3 plate can also accommodate two BioCells and one standard spectrophotometric cuvette. BioCell (also from BioTek) is a 1 cm pathlength vessel that requires less sample volume than a standard 3 mL cuvette. All 1 cm pathlength measurements were made with BioCell.
Herring sperm DNA, BCA kit, bovine serum albumin, and all buffer salts were obtained from Sigma-Aldrich. The Vybrant® MTT Cell Proliferation Assay Kit was from Invitrogen, part of Life Technologies. Primary mesothelioma cells and thiostrepton were gifts from Professor Nick Heinz at University of Vermont. Standard, black polystyrene 96-well microplates used in the cytotoxicity assay were from Corning.
Double-stranded DNA (dsDNA) standards were created by serial dilution of a concentrated herring sperm DNA stock in TE buffer (10 mm TRIS, 1 mm EDTA, pH=7.0). Take3 microvolume data was obtained with undiluted standard samples. BioCell data was acquired using either undiluted or, for higher concentration samples, a 20-fold dilution of standard in TE buffer. All sample measurements at 260 nm were background corrected using a TE buffer blank at 320 nm. All concentrations depicted are based on a 1 cm pathlength and 50 ng/µL OD.
A method was developed to allow in situ analysis directly on the Take3 plate microspots. Briefly, the BCA assay was made by adding 2 µL protein standards and samples, followed by 2 µL BCA working reagent directly onto the microspots.
Protein standards and samples in duplicate were added with a single channel pipette followed by addition of BCA working reagent using a multichannel pipette with mixing. Incubations were performed at room temperature for 25 minutes. Curve fits used quadratic functions as recommended by kit protocols. All BCA assay measurements were made at 562 nm and blank corrected. The blank was composed of a 1:1 volume ratio of BCA working reagent and de-ionized water.
Primary human mesothelioma cells were seeded at 20,000 cells per well in 200 µL of DMEM/F12 media supplemented with 10% FBS, and were incubated overnight at 37°C in a humidified 5% CO2 environment. The cells were then treated with thiostrepton, a polypeptide antibiotic, at concentrations ranging from 20–0.0195 µM with eight replicates per concentration and incubated for an additional 24 hours. MTT reagent was then added, and absorbance was read at 570 nm. All absorbance measurements were blank corrected where the blank consisted of media and MTT reagent.