384-well plates reading

However, many recent instruments are still not considered satisfactory, since professional developers in the field of high-throughput screening (HTS) want to use the full performance of the latest generation of robots and computers for automation. This results in new instrumental developments, like the possibility of reading not only 96, but 384 or even 1536 wells plates as well as DNA chips, very rapidly (in a minute or so) and repeatedly without any mechanical failures. Hence, in the eyes of company scientists developing new assays, many present-day instruments still correspond to an intermediate stage of development. For research laboratory scientists, on the other hand, the actual equipment offers excellent performance. 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

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. 

Stop the reaction with equal volumes of stop solution added to each well before reading the plates 1% SDS is the stop solution for the ABTS substrate, and 5% EDTA is the stop solution for p-NPP substrate. 

All determinations are performed in duplicate. The blank is 10 pi BSA solution + 10 pi MU-NeuAc substrate pH 4.3 in a well of a 96-well plate. The sample comprises 10 pi homogenate (diluted until the protein concentration is 3 mg/ ml) + 10 pi of substrate. Incubate the samples for 1 h at 37°C on a heat block. Add 200 pi carbonate buffer and read the fluorescence. For calibration, 25 pi 4-MU standard solution (750 pmol) is mixed with 200 pi carbonate buffer and measured. For / -galaclosidase, the homogenate is diluted until the protein concentration is 0.3 mg/ ml. The incubation solution is as follows 10 pi + 20 pi MU-Gal. 

Ten milligrams of 2,2 -azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt is dissolved in 50 ml of substrate buffer containing 10 pi of 30% hydrogen peroxide. Only freshly prepared substrate should be used. Incubation is carried out for 30 min at 37°C, followed by visual plate reading on a 1+ to 4+ basis or at 540 pm if a microtiter plate reader is available. The optical density of the instrument is set to zero with the antigen control (well 12 in the first row). Any optical density in the antibody controls (bottom row) is subtracted from the corresponding test well above. 

The 8X12 library was simply irradiated with a hand-held UV lamp (365 nm) to discriminate easily fluorescent and nonfluorescent polymers and to visualize the corresponding emission color in solution. Then, with a spectrofluorimeter able to read 96-well plates for several excitation and emission wavelength combinations, the different excitation wavelengths were evaluated. By this procedure, new polymers showing green (39a,b, excitation at 460 nm, emission detection at 530 nm) or blue (39c-e, excitation at 360 nm, emission detection at 460 nm) emitting fluorescence were rapidly discovered (Fig. 5.13). 

As discussed previously, read time is often a bottleneck in the HCS process and will dictate upstream liquid handling processes unless a pause step (as with fixed cells) is included. Assays with many 384-well plates in live cell mode become difficult, even with a fully integrated robotic system, because the cell growth, cell plating, compound addition, and other steps require precise coordination with imaging that is often not worth the cost. 

Color development in the substrate wells can be quantitated by spectrophotometry or, less precisely, by eye. If spectrophotometry is to be done directly in the Microtiter plate wells, plates with flat-bottomed wells are used and the beads are removed so as to allow an unobstructed beam of light to pass through the colored substrate in each well (as with the Flow Laboratories plate reader). The standard curve principle can be used, as for RIA quantitation. If the human eye is used for reading color... 

Dry the outsides of the plates with paper to help avoid water stains, and then dry the plates at 37°C. When dry, add 100 pi well of 33% glacial acetic acid (33 ml 100 mH) (BDH) and mix the contents of each well before reading at 570 nm. 

Transfer 100 pL of the supernatants from each well to designated wells into a clean 96-well plate, and read at 405 nm using an available ELISA plate reader. 

Quantify the luciferase activity using a commercial kit Luciferase assay system (PROMEGA) Load 10 pL of the lysed cells into a 96-well plate and place it into the lecture plate reader. Load the luciferase substrate to the injector. 50 pL of the luciferase substrate is injected via an injector, and the absorbance is read immediately at 563 nm on a Wallac Victor2 1420 Multilabel Counter (Perkin Elmer). 

Martin et al. published a paper on the theoretical limits of HPLC which is well worth reading.They used relatively simple mathematics to calculate pressure-optimized columns for which the length L, particle size and flow rate u of the mobile phase were selected such that a minimum pressure Ap is required to solve a separation problem. It has been shown that these optimized colunms are operated at their van Deemter curve minima. Some astonishing facts have emerged from the study, provided that the chromatography is performed on well packed columns (reduced plate height h = 2-3 see Section 8.5). 

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