Light scattering assays

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. 

Four methods of quantitating apoptosis by flow cytometry are described in this chapter. The two-dimensional light scatter assay measures the reduction in cellular volume and increase in cell density that are observed during apoptosis and that are revealed by a decrease in forward light scatter and an increase in... 

Fig. I. Two-dimensional light scatter assay. Burkitt lymphoma cells induced into apoptosis by incubation with a MAb against IgM were assayed for forward light scatter (FSC) and 90° light scatter (SSC). Region 1, viable cells Region 2, apoptotic cells.

The two-dimensional light scatter assay can be equally well carried out on viable cells. 

Another important limitation to be aware of is that the induction of apoptosis in a population of cells will not always result in a clearly distinct subdiploid peak (12), We have seen this particularly when apoptosis is induced in Burkitt lymphoma cells by serum deprivation. After approx 2 wk, all cells are apoptotic as can be clearly seen by the two-dimensional light scatter assay and confirmed by microscopic analysis of acridine orange-stained cells, but the cell-cycle profile gives the appearance of a mainly viable population (Fig. 5). 

Fig. 5. Comparison of two-dimensional light scatter assay with subdiploid DNA peak assay. Burkitt lymphoma cells were serum-deprived for 14 d and then assayed for (A) forward light scatter (FSC) versus 90° light scatter (SSC) or (B) cell cycle. Note that although all the cells are clearly apoptotic as shown by the light scatter assay, there is only a small subdiploid peak (SD) present in the cell-cycle analysis, demonstrating that in this case, celt-cycle analysis alone would grossly underestimate the extent of apoptosis present in the cell population.

Light-scattering assay for aggregates nonionic detergent [53]... 

Figure 7. Species-specific dissolution of isolated VEs by purified lysins as determined by the light scattering assay. Vertical axis, percent VE dissolved horizontal axis, qg lysin added. R j, VEs from the red abalone, H. rufescens. B j, VEs from the black abalone, H. cracherodii. Pyj, VEs from the pink abalone, H. corrugata. ( ) red lysin (A) pink lysin and ( ) black lysin (from Vacquier and Lee, 1993).

Fig. 2. Light scattering assay of the assembly of COPII coat with Secl2ACp and GTP. (A) The light scattering of a suspension of major-minor liposomes (100 ml ) in HKM buffer...

It is important to note that the fluorescence and light scattering assays are non-invasive techniques and thus can be coupled to other techniques such as biochemical fractionation or electron microscopy. The sample size and concentration of reagents are in the same range as those used in other techniques, thus facilitating comparison. 

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