Multicolor immunofluorescence staining in conjunction with DNA analysis has been limited by the availability of fluorochromes that can be discriminated in a single analysis. In order to obtain the maximum amount of information from a single specimen, a protocol was designed to take advantage of the LSC cell position feature. When a specimen is analyzed on the LSC, the position of each cell is recorded in the .fcs data file. Data files from multiple analyses of a single specimen are merged on a cell-by-cell basis according to the position of each cell. Thus, experiments utilizing dyes with overlapping spectral characteristics are possible by analyzing the specimen multiple times and restaining between scans.

Staining Procedure and LSC Analysis A preparation of ammonium chloride-lysed whole blood was placed in a chamber slide, and the cells allowed to adhere. The cells were stained with CD45-CY5 for 20 minutes in a humidified chamber, then washed with PBS, and analyzed by the LSC utilizing a red HeNe laser. The cells were contoured by red light scatter, and the CD45-CY5 was measured. The same cells were restained with a mixture of CD4-PE, CD19-PE/TR and CD3-PE/CY5, washed and analyzed by the LSC, utilizing the argon laser and contouring on blue light scatter. For the third analysis, the cells were stained with CD8-FITC, then fixed by passing ethanol over the cells to allow DNA staining by propidium iodide. The optical filter blocks were changed for this analysis in order to measure the green FITC signal and the red PI signal. The three data files were merged by the LSC WinCyte™ software into a single data file. Fluorescence compensation was set in the software after merging the data files. (Data are from Dr. Richard Clatch, Department of Pathology, Highland Park Hospital, Highland Park, IL.)

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