Microtiter plates , 384-well

Avdeef and Bucher [24] investigated the use of universal buffers in potentiomet-ric titrations. Recently, such a buffer system, formulated with several of the Good components, has been designed specifically for robotic applications, where automated pH control in 96-well microtiter plates is required, with minimal interference to the UV measurement [48]. This universal buffer has a nearly perfectly linear pH response to additions of standard titrant in the pH 3-10 region [8, 48]. 

Ishihama et al. [147] have describe a rapid screening method for determining pK values of pharmaceutical samples by pressure-assisted CE, coupled with a photodiode array detector. Each CE run was completed in less than 1 min, so a 96-well microtiter plate could be measured in one day. Determinations of the pKa values of 82 drugs illustrated this interesting new method. 

A high-throughput method using a 96-well microtiter plate format and plate UV spectrophotometry has been described [26]. Solubilities at a single pH, or at <12 pH values can be determined, using one of two methods. 

In the commercial version of the PAMPA assay, a sandwich (Fig. 7.9) is formed from a specially-designed 96-well microtiter plate [pION] and a 96-well microfilter plate [several sources], such that each composite well is divided into two chambers donor at the bottom and acceptor at the top, separated by a 125-pm-thick microfilter disk (0.45 pm pores, 70% porosity, 0.3 cm2 cross-sectional area), coated with a 10% wt/vol dodecane solution of egg lecithin (a mixed lipid containing mainly PC, PE, a slight amount of PI, and cholesterol), under conditions that multilamellar bilayers are expected to form inside the filter channels when the system contacts an aqueous buffer solution [543]. 

Four neutral lipid models were explored at pH 7.4 (1) 2% wt/vol DOPC in dode-cane, (2) olive oil, (3) octanol, and (4) dodecane. Table 7.5 lists the effective permeabilities Pe, standard deviations (SDs), and membrane retentions of the 32 probe molecules (Table 7.4). The units of Pe and SD are 10 6 cm/s. Retentions are expressed as mole percentages. Figure 7.22a is a plot of log Pe versus log Kd (octanol-water apparent partition coefficients, pH 7.4) for filters loaded with 2% wt/vol DOPC in dodecane (model 1.0, hlled-circle symbols) and with phospholipid-free dodecane (model 4.0, open-circle symbols). The dashed line in the plot was calculated assuming a UWL permeability (see Section 7.7.6) Pu, 16 x 10-6 cm/s (a typical value in an unstirred 96-well microtiter plate assay), and Pe of 0.8 x 10-6 cm/s... 

An infrequently used method (in pharmaceutical research) for determining the UWL permeability involves measuring transport of molecules across a high-porosity microfilter that is not coated by a lipid. The molecules are able to diffuse freely in the water channels of the microfilter. The filter barrier prevents convective mixing between the donor and acceptor sides, and an UWL forms on each sides of the microfilter. Camenisch et al. [546] measured the effective permeabilities of a series of drug molecules in 96-well microtiter plate-filterplate (Millipore GVHP mixed cellulose ester, 0.22 pm pore) sandwich where the filters were not coated by a lipid. The permeabilities were nearly the same for all the molecules, as shown in Fig. 7.8a. Our analysis of their data, Fig. 7.8b, indicates / aq = 460 pm (sandwich stirred at 150 rpm). We have been able to confirm similar results in our laboratory with different microfilters, using the lipid-free method. 

A robot that dispenses culture media, solvents, cell suspensions, antimicrobial agents, or other liquid reagents into 96-well microtiter plates. 

He et al. (2002) used an off-line HPLC/CE method to map cancer cell extracts. Frozen ovarian cancer cells (containing 107 cells) were reconstituted in 300 pL of deionized water and placed in an ultrasonic bath to lyse the cells. Then the suspension was centrifuged and the solubilized proteins were collected for HPLC fractionation. The HPLC separation was carried out on an instrument equipped with a RP C-4 column, 250 mm x 4.6 mm, packed with 5-pm spherical silica particles. Extracted proteins were dissolved in 300 pL of DI water, and lOOpL was injected onto the column at a flow rate of 1 mL/min. Buffer A was 0.1% TEA in water and buffer B was 0.1% TFA in acetonitrile. A two-step gradient, 15-30% B in 15 min followed by 30-70% B in 105 min, was used. The column effluent was sampled every minute into a 96-well microtiter plate with the aid of an automatic fraction collector. After collection, the fractions were dried at room temperature under vacuum. The sample in each well was reconstituted before the CE analysis with 10 pL deionized water. The... 

In a different approach three different structurally defined aza-crown ethers were treated with 10 different metal salts in a spatially addressable format in a 96-well microtiter plate, producing 40 catalysts, which were tested in the hydrolysis of /xnitrophenol esters.32 A plate reader was used to assess catalyst activity. A cobalt complex turned out to be the best catalyst. Higher diversity is potentially possible, but this would require an efficient synthetic strategy. This research was extended to include lanthanide-based catalysts in the hydrolysis of phospho-esters of DNA.33... 

It is well known that acrylates undergo transition metal catalyzed reductive aldol reaction, the silanes R3SiH first reacting in a 1,4 manner and the enolsilanes then participating in the actual aldol addition.57,58 A catalytic diastereoselective version was discovered by arrayed catalyst evaluation in which 192 independent catalytic systems were screened on 96-well microtiter plates.59 Conventional GC was used as the assay. A Rh-DuPhos catalyst turned out to be highly diastereoselective, but enantioselectivity was poor.59... 

In a different approach a super-high-throughput ee-assay was developed on the basis of chirally modified capillary array electrophoresis (CAE).90 CAE was used in the Human Genome Project, and commercially available instruments have been developed which comprise a high number of capillaries in parallel, for example the 96-capillary unit MegaBACE consisting of 6 bundles of 16 capillaries.91 The system can address a 96-well microtiter plate. It was adapted to perform ee-determinations of chiral amines, which are potentially accessible by catalytic reductive amination of ketones, transition metal catalyzed Markovnikov addition of ammonia, or enzymatic hydrolysis of acetamides (Scheme 14).90... 

CombiCHEM System (Fig. 3.9) For small-scale combinatorial chemistry applications, this barrel-type rotor is available. It can hold two 24- to 96-well microtiter plates utilizing glass vials (0.5-4 mL) at up to 4 bar at 150 °C. The plates are made of Weflon (graphite-doped Teflon) to ensure uniform heating and are sealed by an inert membrane sheet. Axial rotation of the rotor tumbles the microwell plates to admix the individual samples. Temperature measurement is achieved by means of a fiber-optic probe immersed in the center of the rotor. 

Parallel processing of synthetic operations has been one of the cornerstones of medicinal and high-throughput synthesis for years. In the parallel synthesis of compound libraries, compounds are synthesized using ordered arrays of spatially separated reaction vessels adhering to the traditional one vessel/one compound philosophy. The defined location of the compound in the array provides the structure of the compound. A commonly used format for parallel synthesis is the 96-well microtiter plate, and today combinatorial libraries comprising hundreds to thousands of compounds can be synthesized by parallel synthesis, often in an automated fashion. 

We have developed chemiluminescent immunoenzymatic assays for (3-ago-nist drugs in the 96-well-microtiter-plate format. Such competitive assays have been used for determination of clenbuterol and of the overall content of p-agonist drugs in the sample. They matched the standard requirements of precision and accuracy, and were more sensitive compared to the conventional colorimetric methods. Moreover, CL detection was very rapid, making these assays suitable for screening analysis. 

Although other assay containers can be used (e.g., coated tubes or beads), the most popular one is the 96-well microtiter plate. ELISAs that are detected in a microplate luminometer must be performed in opaque white or black plates. 

The cell titer of each culture is counted and a sample diluted in CM 10 for assessment of posttreatment survival. For this, two 96-well microtiter plates are charged with 200 pi of a diluted cell suspension, using a multichannel pipette such that each well contains on average one cell. 

For estimation of cloning efficiency and mutant induction, cells are plated out in 96-well microtiter plates. Flasks and microtitre plates are incubated at 37°C in a C02 incubator as in the cytotoxicity assays. 

Fluorometers designed for research purposes(31) are typically equipped with a xenon arc lamp, monochromators, one or more photomultiplier tubes, cuvet holders, and a computer interface. Some research level fluorometers, such as the Perkin-Elmer LS50, have optional microtiter plate reading accessories with fiber optic bundles. This is convenient since 96-well microtiter plates are commonly used for immunoassay development, and many commercial immunoassays are based on the use of microtiter plates. Fluorometers designed for commercial immunoassay purposes are generally dedicated instruments with few, if any, data acquisition and reduction parameters that can be manipulated by the user. 

The use of 96-well microtiter plates coupled with the rapid quantification by a spectrophotometric plate reader allows this system to screen a large array of compounds in a relatively short period of time. Alternatively, the compound analysis can be performed with fast and sensitive LC/MS by pooling the samples together. Both the spectrophotometric and mass spectrometric methods... 

Schnizler K, Kuster M, Methfessel C, Fejtl M. 2003. The roboocyte Automated cDNA/mRNA injection and subsequent TEVG recording on Xenopus oocytes in 96-well microtiter plates. Receptors Channels 9 41. 

Figure 6 Lipofection results (lipofection profiles) of lipoplexes from the R-configu-rated cationic lipids KL-1-1 to KL-1-17 (Table 1) in a mixture with equimolar amounts of l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (counterion chloride) and the pCMVluc-plasmid. Each bar represents the mean ( S.D.) of three wells of a 96-well microtiter plate. T-axis (left) represents the transfection efficiencies expressed in relative light units (RLU) (lu/pg protein). X-axis (right) represents the viability of the cells compared to nontreated control cells. F-axis represents the different cationic lipid/plasmid DNA-charge ratios from 1 to 15.

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