Microtitre plate methods

Lucas M, Mertens V, Corbisier A-M, Vanhulle S (2008) Synthetic dyes decolourisation by white-rot fungi development of original microtitre plate method and screening. Enzyme Microb Technol 42 97-106... 

Bevan, C. D. Lloyd, R. S., A high-throughput screening method for the determination of aqueous drug solubility using laser nephelometry in microtitre plates, Anal. Chem. 72, 1781-1787 (2000). 

Hence, this assay is an extremely useful and selective assay to measure O2 secretion. Because of this selectivity and because it measures the initial product of O2 reduction, it is often used as the method of choice to detect NADPH oxidase activity. It is suitable for semi-automation because assays can be performed in 96-well microtitre plates (using ELISA plate readers with a suitable filter), or cytochrome c reduction can be detected using simple spectrophotometers. The assay, however, is not suitable for measuring O2 that may be generated intracellularly within activated neutrophils. 

Incubate at least four series, cells with three or more different concentrations of the preparation to be examined and the reference preparation in a microtitre plate and include in each series appropriate controls of untreated cells. Choose the concentrations of the preparations such that the lowest concentration produces some protection and the largest concentration produces less than maximal protection against the viral cytopathic effect. At a suitable time add the cytopathic virus to the wells with the exception of a sulScient number of wells in ah series, which are left with uninfected control cells. Determine the cytopathic effect of virus quantitatively with a suitable method. Calculate the potency of the preparation to be examined by the usual statistical methods for a parallel line assay. 

Murray AM, Kelly CD, Nussey SS, Johnstone AP. (1998) Production of glutathione-coated microtitre plates for capturing recombinant glutathione S-transferase fusion proteins as antigens in immunoassays. J Immunol Methods 218, 133-9. 

Price, M. R., Sekowski, M., Hooi, D. S. W., Durrant, L. G., Hudecz, F., and Tendler, S. J. B. (1993) Measurement of antibody binding to antigenic peptides conjugated in situ to albumin-coated microtitre plates. J. Immunol. Methods 159, 277—281. 

Das Sarma, Duttagupta C, Ali E, et al. Direct microtitre plate enzyme immunoassay of folic acid without heat dena-turation of serum. /. Immunol. Methods (1995) 184 7-14. 

An alternative method which can also be automated by the use of the Titertek supernatant harvester (see Appendix 3) involves the measurement of radioactive chromium released into the culture medium from killed cells. The harvester consists of a set of absorbent cylinders aligned so that they may be inserted into the wells of a microtitration plate (Appendix 3). Once the supernatant in the wells has been absorbed the cylinders are transferred to counting vials and the amount of radioactive chromium released from the cell monolayer is estimated. Cells take up 51 Cr sodium chromate rapidly and the excess is readily washed away by rinsing in culture medium. 

For those interested in cloning reference should be made to Chapter 7. A small scale perfusion vessel is considered in 3.4.3. For many biochemical studies involving incubation of cells with radioisotopes in the presence of drugs, anti-metabolites, hormones etc. small numbers of cells are required and these may conveniently be grown on the bottoms of glass scintillation vials or in the wells of a 6 or 24 well TC plate or even in the wells of a microtitre plate (see Table 3.1). This last method enables 96 replicate cultures to be handled simultaneously but the maximum volume that each well will hold is 0.25 ml. 

This is a method of measuring total nucleic acid which is equivalent to cell number. The method described is for use with cells growing in microtitre plates but it can readily be scaled up. 

A sandwich ELISA is used to search for a desired analyte in a test solution, as follows the solid phase is coated with analyte-specific capture antibodies to pull the analyte out of the test sample. After washing, the amount of analyte bound to the solid phase can be determined by adding an excess of enzyme-labelled analyte-specific antibody. The specificity of the method can be improved by using a sandwich-type, two-site assay, in which the capture and labelled antibodies have specificities for different parts of the analyte, as mentioned above. The performance of an immunoassay in a standard microtitre plate requires several hours. Such long incubation times are mostly linked to inefficient mass transport from the solution to the surface, whereas the immunocapture itself is a rapid process. 

Immunochemical methods for low molecular weight (<1000 D) environmental compounds, such as pesticides, nitro aromatics, and PAHs (polycyclic aromatic hydrocarbons) have been developed since more than 25 years (e.g. Hammock and Mumma, 1980 Hammock et al., 1990 Dankwardt, 2000). The most common method is the immunoassay, which is either carried out with (heterogeneous) or without (homogenous) separation steps. Heterogeneous formats that use microtitre plates, plastic tubes and/or membranes as solid supports are more commonly used. 

The activity of cholinesterases is usually determined by Ellman s method (19). This assay can also be carried out in microtitre plates. Thirty microliters of the ChE dilution and 30 pL of DNTB are added to 210pL of40mM Britton-Robinson-I buffer pH 8.0. The reaction is started by addition of 30 pL of the appropriate acylthiocholine substrate. A microtiter plate reader is used to monitor the development of the absorption at 595 nm. The corresponding slope is used to calculate the esteratic activity. 

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