Light scattering procedure

The light scatter assay may be used to determine absolute numbers of viable cells if flow cytometry data from cell suspensions of known concentration are used to construct a standard curve. For that purpose, cell concentrations should be determined in a series of graded, standard cell suspensions with the use of a Coulter counter. A plot of those standard concentrations versus the number of events (Hght scatter signals) acquired during a specified acquisition interval in the flow cytometer may then be used to interpolate cell concentrations for test samples that have been assayed by the light scatter procedure. 

Traditionally, low concentrations of tag (i.e., deuteriated) molecules have been used in neutron scattering. This practice was a continuation of light scattering procedures, where it was necessary to extrapolate to zero concentrations to obtain meaningful results because of interactions between the polymer and solvent. However, if the only difference between a tagged molecule and matrix is the degree of deuteriation the necessity of using low tag concentrations is removed. 

This chapter is the narrowest in scope of any chapter in this book. In it we discuss a single experimental procedure and its interpretation. It is appropriate to examine light scattering in considerable detail, since the theory underlying this method is relatively unfamiliar to students and the interpretation yields information concerning a variety of polymer parameters. 

Until now we have looked at various aspects of light scattering under several limiting conditions, specifically, C2 = 0, 0 = 0, or both. Actual measurements, however, are made at finite values of both C2 and 6. In the next section we shall consider a method of treating experimental data that consolidates all of the various extrapolations into one graphical procedure. 

Seligson s group (95) has published a similar turbidimetric procedure but used nephelometry to measure continuously the effect of lipase on the light scattering of an olive oil emulsion. The instrumentation and approach is the same as that described above for the nephelometric determination of amylase. The method according to the authors is fast and precise with good specificity and sensitivity. The short time required for analysis makes it suitable for emergency use. The technical simplicity permits this method to be easily automated, and it appears to be the lipase method of choice. 

Abstract Flow cytometry is a technique for rapidly examining multiple characteristics of individual cells, by recording fluorescence signals emitted from cell-associated reporter molecules, and measuring cellular light scattering properties. This chapter introduces the principles and practice of flow cytometry, and reviews examples from the literature that highlight applications of this experimental tool in the neurosciences. The chapter concludes with protocols for three basic procedures that illustrate some practical aspects of analytical flow cytometry. 

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