Cassette incubation

Weaver, R. et al. 2003. Cytochrome P450 inhibition using recombinant proteins and mass spectrom-etry/multiple reaction monitoring technology in a cassette incubation. Drug Metab. Dispos. 31 955. 

Delete the lox-STOP-lox cassette in vitro by incubating 1 pg conditional shRNA plasmid DNA with 4 units of Cre recom-binase (New England Biolabs) in a total volume of 50 pi lx Cre buffer at 37°C for 30 min. Inactivate the recombinase for 10 min at 70°C. Eliminate nonrecombined plasmids by EcoRI digestion (see Note 8). 

Substitute Soln. B by the AMPPD dilution (0.2 ml/cm ) and incubate for 5 min. Drain the blot and wrap it with a solvent-tight foil. Incubate the wrapped membrane at 37 °C for 5 min, and then expose the blot face towards to the film in an X-ray film cassette for 15 min to 24 h or photograph it with a digital camera. 

Drags are tested at 10 p,M concentration and in two directions (apical to basolateral (a-b) and basolateral to apical (b-a)). Monolayer efflux studies are conducted at 37 °C in a humidified incubator with shaking (90 rpm) for 60 min. Transendothelial electrical resistance is measured with an Endohm Meter (World Precision Instruments, New Haven, CT). Reference drags for paracellular transport (14C-mannitol), tran-scellular transport (3H-propranolol), and Pgp efflux (amprenavir) should be included in each experiment. Concentrations of 14C-mannitol and 3H-propranolol are measured by liquid scintiallation counting. Amprenavir is analyzed by cassette LC/MS/MS analysis along with the test drags. 

Special hybridization ovens or convenient acrylic cassettes to be placed in a waterbath are commercially available. The membrane is placed in the hybridization cassette (e.g., from Bios Corp., New Haven, CT 06511 Fax (203) 773-0017), covered with (pre)hybridiza-tion solution, the top closed over a cover and incubated at the desired temperature. Hybriboxes can also be constructed as reported by Cohen et at. (1990 Section 7.2.6). The hybridization... 

Before prehybridization, nitrocellulose should be wetted with 1 X SSPE (or 1 X SSC), containing 0.1% SDS, whereas nylon membranes can be used directly. Prehybridization and hybridization is performed in polythene bags, plastic boxes (hybridization cassette) or hybridization incubators (Section 8.1.2). The cassettes and incubators are economical in the use of hybridization solutions. For plastic bags, use 0.25 ml/cm for nylon membranes and 0.1 ml/cm for nitrocellulose membranes of prefiltered (important ) prehybridization solutions. The bags should be massaged to completely distribute the solution and incubated with agitation. 

At the end of incubation, set up the Combi dispenser with plastic tip cassette, and dispense 1.5 pi ColorLock Gold working solution to all occupied wells in the 1,536-well assay plate see Note 3). Spin down the plate and wait 5 min before dispensing 1 pi stabilizer working solution with a different plastic tip cassette to all occupied wells. Spin down again and read on the PHERAstar plate reader under OD635. Seal the plate to prevent evaporation if the reader is currendy unavailable. 

With the Multidrop Combi dispenser and small metal tip cassette, dispense 3 pi Assay Buffer in columns 1-2 for Positive Control, and 3 pi diluted ENPPl in columns 3 8. With a different cassette, dispense 3 pi diluted ATP solution to all assay wells. Spin down the plate and seal with aluminum adhesive seal. Incubate for 110 min. 

Free (wash) solution was removed from the filter with filter paper, positions A12 and HI marked with ball point (not a felt pen ). The filter was placed in a closely fitting box, 10 ml of substrate solution was added and incubated for 1 min. The filter was wrapped, straight from the substrate in plastic, without air bubbles. Liquid pressed out, the filter transferred in plastic into the luminometer cassette and placed in the luminometer. Luminescence was measured according to the required program. Data were collected in a file and for each sample the level of N7-HETE-dG/107 nucleotides was calculated (in an Excel worksheet). 

The visualization of hybridization between the RNA probe and the DNA fragments on the blot is based on an enzyme-linked immunoassay. An antidigoxigenin/alkaline phosphatase conjugate is bound to the digoxigenin component of the probe and then incubated with the substrates X-phosphate and nitroblue tetrazolium (see Section 2.) under alkaline conditions, which will result in purple-blue precipitates on the membrane within a couple of hours. Color detection thus saves time and money as it does not require X-ray films, cassettes, and intensifier screens. For a permanent record the result can be photocopied or photographed. 

One recent example of a high-throughput assay for metabolic stability was reported by Fonsi et al. (2008). In this report, the authors described the use of a robotic platform to prepare the compounds for a microsomal stability assay. The authors selected five time points (20, 30, 45, 60, and 90 min) so the intrinsic clearance (CL nt) could be calculated with an Excel spreadsheet. The assay was performed with a triple quadrupole tandem mass spectrometer (MS/MS) system. The system included software tools for automated MS/MS method development—an important requirement for any MS/MS system that will be used for high-throughput assays for microsomal stability assays. The authors also made use of a cassette assay system where samples were pooled after the incubation step was completed. Up to four analytes were pooled and were assayed in one HPLC—MS/MS procedure with a generic chromatographic gradient system. 

Figure 21.8. Standard probe inhibitiors for CYP1A2, CYP 2C8, CYP 2C9, CYP 2C19, CYP 2D6, and CYP 3A4 were incubated at eight inhibitor concentrations and analyzed by LC/MS/MS using cassette analysis, as described in the previous figure. Experimentally derived IC50 values for each of the probe inhibitors compared favorably with Uteratuie values.

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