Paul K. Horan

Automated fluorecent analysis for cytotoxicity assays

Classical measurements of cytotoxicity using dye exclusion and microscopic evaluation are both time-consuming and inaccurate. Using a cell sorter (TPS) a single dye system has been developed which stains live and complement killed cells with different fluorescence intensity. After exposure of target cells to antibody and complement, ethidium bromide is added to the target cells at a high enough concentration to stain complement killed cells very intensely. The cells are then diluted and lysed to produce single nuclei, permitting live cells to be stained but less intensely than the dead cells. Fluorescence intensity is measured on single nuclei at a rate of 10,000 per minute. For these studies anti-T antisera was titrated for complement dependent cytotoxic activity using normal mouse spleen cells, spleen cells from an anti-thymocyte serum treated mouse, and nylon wool purified mouse splenic T-cells. This procedure makes possible, reliable and reproducible measurement of cytotoxic activity on 5,000 cells per determination.

A flow cytometric analysis of the embryonic origin of lymphocytes in diploid/triploid chimeric Xenopus laevis

Quantitative flow cytometry was used to examine the embryonic origin of lymphocytes in Xenopus laevis. Reciprocal head/body transplants were made between diploid (2N) and triploid (3N) embryos of the same developmental stages ranging from neural plate to tail bud stages. Thymuses and spleens were removed from postmetamorphic chimeras. Cell suspensions were stained with the fluorescent DNA stain, mithramycin, and the ploidy (relative fluorescence intensity) of the cells was then determined by flow cytometry. All lymphocytes in the chimeras were derived from the posterior portion of the embryo. In other experiments, various regions of the lateral plate or ventral mesoderm were grafted from triploid to diploid embryos. Only transplants that included middorsal mesoderm gave rise to lymphocytes.

Cellular Proliferation History by Fluorescent Analysis

A variety of biochemical and cellular assays have been developed to make it possible to distinguish between dividing and non-dividing cells (Darzynkiewicz, Z., 1984, and Darzynkiewicz, Z., Traganos, F., and Kimmel, M., 1986, and Dolbeare, F., Beisker, W., Pallavicini, M.G., Banderlaan, M., and Gray, J.W., 1985, and Crissman, H.A., Steinkamp, J.A., 1987, and Muirhead, K.A., Horan, P.K., and Poste, G., 1985). Most of these assays can only determine the state of proliferative activity at the time the cells are “harvested” and do not permit the observer to determine the activity at times prior to harvesting. For example, the elegant methods developed by Darzynkiewicz, et al. (Darzynkiewicz, Z, 1984, and Darzynkiewicz, Z., Traganos, F. and Kimmel, M., 1986) permit the investigator to distinguish cycling from non-cycling cells at the time of harvest using dual-fluorescence measurements of DNA and RNA levels, but they do not permit evaluation of the proliferative activity of these same cells at times prior to harvesting. In this manuscript, a novel method of labeling cells is presented which does not affect the viability or proliferative activity of cells, but permits retrospective monitoring of their growth for as many as eight cell divisions. The dye, PKH-1 (Zynaxis Cell Science, Inc., 371 Phoenixville Pike, Malvern, PA USA 19355), is applied to the cells and results in uniform staining of the plasma membrane. The cellular growth rate is unaffected by the presence of the dye. Further, the dye does not leak out of the cells and it is not transferred from cell to cell within the same culture. When a cell divides, each resulting daughter cell receives one-half of the total dye present in the parent cells membrane and each fluoresces at one-half the intensity of the parent cell.