Hts 7000 bio assay reader

UV-visible spectroscopy was performed with an Agilent Hewlett-Packard 8453 diode-array spectrophotometer equipped with an Agilent 89090A thermostat. Absorbance and fluorescence measurements of 96 well plates were accomplished with a Perkin-Elmer HTS 7000 Bio Assay reader. Cells were counted using a Beckman Coulter Z-2 cell and particle counter. Cell viability and angiogenesis was monitored using a Nikon eclipse TS-100 inverted microscope. An Eppendorf thermocycler was used to prepare cDNA. Sequence detection was then carried out using 7300 Real-Time PCR System from Applied Biosystems. An iBlot^® gel transfer device from Invitrogen, Carlsbad, CA was used for dry transfer in Western blots. DNA damage was assessed using a Comet Assay^® Electrophoresis System (Trevigen Inc. Gaithersburg, MD).

Blood samples were collected at each semiannual visit for laboratory testing. Genomic DNA was isolated from peripheral blood leucocytes using the Pure-gene DNA isolation kit (Gentra Systems, Minneapolis, MN). The PICO Green dsDNA quantification kit (Molecular Probes, Eugene, OR) and the Perkin Elmer HTS7000 BioAssay Reader were used for DNA quantification and reading the microplate, respectively. All samples were stored at −20C [36 (link)].

Sup-B15 and FLS cell seeding densities were optimized to ensure cells linearly responded to MTS (Figure S19 and Figure S20, Supporting Information). Sup-B15 cells were then seeded in 24-well glass-bottom plates at a density of 1.7×10⁶ cells/mL and allowed to settle for 1 h, while primary FLS cells were plated at a density of 1×10⁴ cells/mL and allowed to adhere overnight. Cells were then treated with 100 μL of RBCs-(1) at 50% hematocrit (4.45 × 10⁹ cells/mL), or 100 μL of RBCs-(2) at 50% hematocrit (4.3 × 10⁹ cells/mL), or 500 μL free Dex at 2 μM, or 500 μL plain DMEM which was added to wells in Millicell Hanging Cell Culture Inserts (1 μm, polyethylene terephthalate, Millipore). Samples were exposed to a 660 nm LED board for 30 min at RT. After 30 min of exposure, hanging wells were removed and cells were incubated for 24 h at 37 °C in a humidified environment at 5% CO₂. Cells were then treated with 100 μL MTS/1 mL media for 2 h (Abcam 197010) after which absorption at 492 nm was measured using a HTS 7000 BioAssay Reader (Perkin Elmer, Waltham, Ma).

Four millimolars of 4-nitrophenyl-α-D-glucopyranoside (pNPG) was used as a substrate while 50 µg/mL Acarbose served as a positive control. Ninety six-well bioassay microplates were prepared to contain 115 µL of GPE fraction/sub-fraction or control, 90 µL of enzyme solution and 45 µL of substrate solution per well. Absorbance was obtained at a 405 nm wavelength at the start of the reaction and following 30 minutes of incubation at 37 °C, using a Perkin Elmer HTS 7000 Bio Assay Reader and software. Per cent inhibition by tested samples was calculated using the following formula:
$$\%\text{ Inhibition}=100-[({\text{Abs}}_{\text{sample}}/{\text{Abs}}_{\text{control}})\times 100]$$

Cell survival assays based on DNA content were performed in A549 and H596 NSCLC cells as described.^{[18 (link)]} Original H596 cells contain a homozygous *2 NQO1 polymorphism and thereby lack NQO1 expression. Genetically matched NQO1+ counterparts were generated and characterized for responses to β-lap alone as described.^{[20 (link)]} A549 cells endogenously over-express NQO1, and its enzyme activity can be blocked by co-administration of dicoumarol, simulating an NQO1-deficient cell. Briefly, NQO1+ or NQO1− H596 or A549 NSCLC cells were seeded (10,000 cells/well) into each well of 48-well plates. A549 cells were seeded similarly. On the following day, media were removed, and replaced with that containing predetermined doses of free β-lap drug (dissolved in DMSO) or dC₃ micelles with or without PLE for 2 h. For A549 cells, dicoumarol at a concentration of 50 µM was coadministered to inhibit NQO1. After 2 h exposures, media were replaced with control growth media and cells were allowed to grow for an additional 7 days. DNA content was determined by Hoescht dye 33258, using an adaptation of the method of Labarca and Paigen.^{[21 (link)]} Samples were read in a Perkin Elmer HTS 7000 Bio Assay Reader (Waltham, MA) and data were expressed as means±SE relative growth and graphed as treated/control (T/C) values from six wells per treatment.