Cutoff fluorescence selection

Fig. 6.3. Cutoff fluorescence selection for screening. Instrumentation, labeling, and biological noise introduce spreading into a fluorescence measurement, such that the fluorescence probability distributions for wild-type and mutant cells overlap. The logarithm of single-cell fluorescence as measured by flow cytometry is generally well-approximated by a symmetrical Gaussian curve. A cutoff fluorescence value is selected for screening, with all cells above that value sorted out. The enrichment factor forthe mutants is the ratio of (dotted + striped areas)/(striped area), and the probability of retention of a given mutant clone at a single pass is the (striped + dotted area)/(all area under mutant curve).

Fig. 6.4. Yield vs. purity in cutoff selection. For the distributions represented in Fig. 6.3, retention probability (solid line) and enrichment factor (dotted line) were calculated as a function of the cutoff fluorescence value. For example, a cutoff value of 1.4 gives 95 % probability of retention of a given mutant cell, but a fairly modest enrichment factor of 11.8-fold. A cutoffof 3.0 increases the enrichment factor two orders of magnitude to 1000 x, at the cost of an increase in probability of clone loss to 50%.

One-third of these appeared in the actives for the assay, which was defined using a 30% inhibition threshold. The overall hit rate for the assay was 2.0% in other words, 20% of the actives were false positives due to extreme optical interference with the assay readout. This number rises depending on the level of initial fluorescence used as a cutoff in the selection scheme used by the project team at the time, almost half of the actives were rejected as false positives on the basis of the initial fluorescence readings. The corporate screening collection has been reformatted since then, and many of these problem compounds have been removed. 

For normal fluorescence scanning, a high-intensity xenon continuum source or a mercury vapor hne source is used, and a cutoff Alter is placed between the plate and detector to block the exciting UV radiation and transmit the visible emitted fluorescence. For fluorescence measurement in the reversed-beam mode, a monochromatic Alter is placed between the source and plate and the monochromator between the plate and detector. In this mode, the monochromator selects the emission wavelength, rather than the excitation wavelength as in the normal mode. 

Using the calculated values for % binding, a cutoff of 50% of the maximal fluorescence response (compared to 100% for the positive control) is used to select hits for follow-up testing (see Note 50). 

Figure 1 A diagram of the optical arrangement of a stopped-flow system capable of simultaneous observation of changes in absorbance and fluorescence. The light from the xenon lamp is diffracted by monochromator 1 (MCMl) to select the excitation wavelength. Usually quartz optical fibres conduct the light to the observation cell and absorption is detected at 180° and fluorescence emission (wavelength selected by a cutoff filter or MCM2) Is detected at 90° relative to the incident light...

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