Discriminating between damaged and intact cells in fixed flow cytometric samples

A vital, nucleic acid stain (LDS-751) was used to discriminate intact from damaged cells in a flow cytometer even after the samples had been fixed with paraformaldehyde. Three major cell populations with different fluorescence properties with LDS-751 were found in the fixed samples. Cells not staining or only dimly staining with LDS-751 were identified as erythrocytes and platelets, respectively. Cells staining with intermediate amounts of LDS-751 were found to be intact cells, while cells intensively stained were identified as damaged cells. Confirmation of the identity of the populations was obtained by light microscopic examination of the sorted populations and by correlating the fluorescent signals of FDA and LDS-751 in nonfixed cell preparations. Agglutinated cells could also be identified by the increased fluorescent signal in the LDS-751 channel as compared with single cells. The spectral properties of this dye permit excitation at 488 nm with emission in the far red portion of the spectrum. This allowed two-color immunofluorescence to be combined with the intact/damaged cell discrimination on fixed samples. Therefore, intact single cells could be distinguished during flow cytometric analysis, increasing the accuracy of the immunofluorescence measurements. The visualization of the multidimensional data was facilitated using color to discriminate cell populations depicted in multiple perspectives.