Anita P. Stevenson

The Effect of Low-dose Irradiation on Unstimulated and PHA-stimulated Human Lymphocyte Subsets

A culture system was used to evaluate the radiosensitivity of CD4+ and CD8+ T cells, Leu 19+ cells, and B cells obtained from normal adult males. Unstimulated CD8+ lymphocytes (D0 = 55 cGy) were twice as radiosensitive as CD4+ cells (D0 = 115 cGy). B cells had an intermediate radiosensitivity (D0 = 100 cGy). Leu 19+ cells were much more radioresistant and expressed a D0 of 550 cGy. When lymphoid cells were irradiated 1 or 4 days before phytohemagglutinin (PHA) stimulation, they were more radiosensitive than if they were first stimulated with PHA and then irradiated. When lymphoid cells were irradiated 1 h after PHA stimulation each lymphocyte subset was characterized by an increase in the D0 to 150 cGy for B cells to 290 cGy for CD4+ cells, and to 240 cGy for CD8+ cells. In contrast, Leu 19+ cells exhibited a decrease in their D0 to 290 cGy after they were stimulated by PHA.

Use of DiO-C5-3 to improve Hoechst 33342 uptake, resolution of DNA content, and survival of CHO cells☆

Chinese hamster cells (line CHO) stained with either 9 microM Hoechst 33342 (HO) alone or in combination with the membrane potential fluorochrome DiO-C5-3 (DiO) were analyzed using uv laser powers between 25 and 500 mW and sorted for determination of survival by a colony formation assay. The combination of HO-DiO increased fluorescence twofold and provided coefficients of variation (CVs) as low as 3.0% under conditions where viability of cells, even at 500 mW excitation, was unaffected. HO-stained cells yielded CVs of about 8.5% and survivals of approximately 90% under similar analytical conditions. At laser powers of 25 mW, CV values for HO-DiO-stained populations were 4.0% compared to 9.4% for HO-stained cells. Results with another membrane potential dye, rhodamine 123 (R 123), in combination with HO showed no improvement compared to HO-stained cells. No preferential, cell cycle phase-specific killing was observed in either the HO- or HO-DiO-stained populations. CVs of human skin diploid fibroblasts stained with HO or with HO-DiO were comparable over the entire laser power range; however, percentage survival was slightly higher for the HO-DiO-stained populations when analyzed and sorted at the higher power (400-500 mW) range. Long-term cultures of sorted CHO-K1 subpopulations, differing in DNA ploidy, were established from HO-DiO-stained cells. Advantages of this new staining procedure include improved DNA content resolution (low CV values) and the potential use of less expensive FCM uv laser systems coupled with less perturbing excitation powers.

Comparison of Five Short‐Term Assays That Measure Nonspecific Cytotoxicity Mediated to Tumor Cells by Activated Macrophages

Five different short term assays (<48 h) used to measure macrophage‐mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: a) three different populations of macrophages; b) four different kinds of target cells; c) two types of radioisotopes; and d) two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were ap‐

Chapter 9 Supravital Cell Staining with Hoechst 33342 and DiOC5(3)

Publisher Summary The Hoechst (HO342) dyes are benzimidazole derivatives that have a high specificity for double-helical DNA and bind preferentially to A-T base regions, but do not intercalate. Hoechst 33258 has been used in mouse chromosome-banding studies. This dye was quenched when bound to bromodeoxyuridine (BrdUrd)-substituted DNA and developed a method for detecting regions of sister chromatid exchange in metaphase chromosomes labeled with BrdUrd. Although the technique has been useful for a number of cell types, unfortunately HO342 can be limited in usefulness in many cell systems by poor cell cycle resolution due to limited uptake of the dye through the viable plasma membrane, dye toxicity, and loss of long-term viability following ultra-violet (UV) excitation and cell sorting. Dye uptake and/or retention are cell type-dependent and many cell types, including line Chinese hamster ovary (CHO) cells are particularly refractory in this regard. The quality of the DNA histogram for CHO cells stained with HO342 alone is extremely poor. Such results are not acceptable when attempting to obtain a high degree of accuracy in sorting cells from specific phases of the cell cycle.

Tumorigenicity Assays in Nude Mice: Analysis of the Implanted Gelatin Sponge Method

Gelatin sponges, preimplanted in nude mice for 10 days, were used for an improved assay for tumorigenicity of cultured cells. Cells inoculated through the skin into such sponges yielded tumors more rapidly and with greater frequency than with newly implanted sponges or into subcutaneous tissue. However, an unexpected loss of cells occurred in the first few days after implantation. This loss may be an important aspect of tumorigenicity assays of all kinds, and is readily studied with the sponge methods described.