Supravital cell staining with Hoechst 33342 and DiOC5(3).
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Biomedical subjects
A P Stevenson
Publications and source records attributed to A P Stevenson.
Quantitation of the host cell infiltration kinetics of the nonimmunogenic colon 26 tumor by multiparameter flow cytometry.
In this study, we examined the kinetics of host cell infiltration into the nonimmunogenic Colon 26 tumor. We found that 1 x 10(4) cells were required to produce tumors in 100% of mice. The vivo doubling time was 42.5 h, and a barely palpable tumor contained 8 x 10(6) cells. No evidence of concomitant immunity was found. The number of host cells infiltrating the in vivo tumors increased at the same rate as the number of tumor cells, but averaged only 22% of total cells. Cycling T lymphocytes were present in the host cell infiltrate of this tumor. In addition, approximately 50% of in vivo Colon 26 cells were Thy-1.2 positive. The observed characteristic of low immunogenicity makes it a useful murine model for studying human malignant tumors.
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Isolation and reactivity of host effectors associated with the manifestation of concomitant tumor immunity.
In this study, we have measured the specific tumoricidal activity of tumor-infiltrating lymphocytes that prevent the growth of secondary tumors in animals harboring progressing primary tumors (concomitant immunity). Since no tumor grows at the challenge site when concomitant immunity is established, tumor cells were inoculated into a preimplanted gelatin sponge whose subsequent solubilization in collagenase permitted the retrieval of leukocytes after tumor challenge. Primary progressing EMT6 tumors were established in normal BALB/c mice and 10 days later they were challenged with a secondary tumor inoculum introduced through a preimplanted gelatin sponge. At 3, 7, and 10 days after the administration of the tumor inoculum challenge, a monodispersed suspension of infiltrating leukocytes was recovered by collagenase digestion of the sponge matrix and tested for cytotoxicity toward EMT6 tumor targets. Cytotoxic T-lymphocytes with tumoricidal activity accumulated at the site of the secondary tumor challenge by 3 days. This antitumor activity was maximal 7 days following challenge and decayed thereafter. Splenic lymphocytes from these animals showed little cytotoxicity. In animals harboring a primary tumor, lymphocytes found in sponges that were not inoculated with tumor cells were not cytotoxic. We interpret these data to indicate that cytotoxic lymphocytes migrate to, and accumulate at the site of the tumor but not at other sites and that peripheral sources of lymphocytes in tumor-bearing animals such as the spleen may not be the best source of effector cells for evaluating the host's immune response to its tumor. The approach described here may also be useful in studying the mechanisms for host control of metastatic disease.
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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.
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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.
Adult↗
Comparison of five short-term assays that measure nonspecific cytotoxicity mediated to tumor cells by activated macrophages.
Five different short term assays (less than 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: three different populations of macrophages; four different kinds of target cells; two types of radioisotopes; and 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 apparent in assays of less than 24 h duration, disappeared when the same kinds of targets were compared in assays of greater than 40 h duration. The results of this study are an important first step toward standardizing the way in which macrophage-mediated, nonspecific cytotoxicity is measured in short-term assays, laboratory to laboratory.
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Simultaneous measurements of macrophage-induced cytostasis and cytotoxicity of EMT6 cells by flow cytometry.
Cytostasis and cytotoxicity as well as survival and growth of EMT6 cells following exposure to tumoricidal macrophages have been examined using multiparameter flow cytometry. Macrophages were resolved from tumor cells, and the number of surviving EMT6 cells at different interaction times was determined by adding, to each cell suspension, fluorescent latex particles the concentration of which was known and counting them along with the cells. The percentages of EMT6 cells in S phase were determined by analysis of DNA histograms and compared to the percentages as determined by autoradiography. The progression of cells through the cell cycle was also examined during exposure to macrophages by dual parameter analysis of DNA content and bromodeoxyuridine incorporated into DNA prior to exposure to macrophages. The results showed that, by 4 h of interaction with tumoricidal macrophages, EMT6 cells stopped progressing; tumor cell progression was inhibited in all phases of the cell cycle. By 24 h, there was an absolute decrease in survival. By 48 h, however, surviving tumor cells which escaped the tumoricidal activity of macrophages exhibited a population doubling rate similar to control cells.
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Potassium ion influx measurements on cultured Chinese hamster cells exposed to 60-hertz electromagnetic fields.
Potassium ion influx was measured by monitoring 42KCl uptake by Chinese hamster ovary (CHO) cells grown in suspension culture and exposed in the culture medium to 60-Hz electromagnetic fields up to 2.85 V/m. In the presence of the field CHO cells exhibited two components of uptake, the same as previously observed for those grown under normal conditions; both these components of influx were decreased when compared to sham-exposed cells. Although decreases were consistently observed in exposed cells when plotted as loge of uptake, the differences between the means of the calculated fluxes of exposed and sham-exposed cells were quite small (on the order of 4-7%). When standard deviations were calculated, there was no significant difference between these means; however, when time-paired uptake data were analyzed, the differences were found to be statistically significant. Cells exposed only to the magnetic field exhibited similar small decreases in influx rates when compared to sham-exposed cells, suggesting that the reduction in K+ uptake could be attributed to the magnetic field. Additionally, intracellular K+ levels were measured over a prolonged exposure period (96 h), and no apparent differences in intracellular K+ levels were observed between field-exposed and sham-exposed cultures. These results indicate that high-strength electric fields have a small effect on the rate of transport of potassium ions but no effect on long-term maintenance of intracellular K+.
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Macrophage-induced cytostasis: kinetic analysis of bromodeoxyuridine-pulsed cells.
The effect of tumoricidal macrophages on the cell cycle progression of six different cell lines was studied using an anti-bromodeoxyuridine (BrdUrd) monoclonal antibody to follow the traverse of BrdUrd-labeled cells. Exponentially growing cultured mammalian cells, from six different cell lines, were prepulsed with BrdUrd before exposure to tumoricidal macrophages. The cultured cells were then analyzed as a function of time for DNA content (by propidium iodide staining) and for BrdUrd incorporation (using a fluoresceinisothiocyanate [FITC]-conjugated anti-BrdUrd monoclonal antibody). The position of the cells in cycle and the progression of the BrdUrd-labeled cohort was followed using flow cytometry. The cell lines examined were: Colon 26, BALB/c-3T3, ST3T3 (a spontaneously transformed, tumorigenic clone of 3T3), WCHE5 (a clone of whole Chinese hamster embryo cells), RIF (a radiation-induced fibrosarcoma), and A101D (a human melanoma). The bivariate distributions showed that for all six cell lines the BrdUrd-labeled cohort in the control cultures progressed around the cell cycle during the first 12 h of culture, as the cells exponentially increased. In contrast, when each cell line was incubated with tumoricidal macrophages, the BrdUrd-labeled cohort did not progress through cell cycle but remained in S phase throughout the 12-h culture period. There was also no evidence for progression of cells out of G1. The data show that cells were arrested in every phase of cell cycle. This study suggests that cytostasis, as manifested by the termination of progression in all phases of the cell cycle, is a universal phenomenon induced by tumoricidal macrophages.
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A gelatin sponge model for studying tumor growth: flow cytometric analysis and quantitation of leukocytes and tumor cells in the EMT6 mouse tumor.
This study examined the recruitment of host cells into a progressing EMT6 tumor following the inoculation of tumorigenic cells into a preimplanted gelatin sponge. Tumor cell proliferation occurred to a greater extent in sponge tumors than in tumors obtained by direct subcutaneous injection of tumor cells. Blank sponges, lacking tumorigenic cells and used as controls, elicited an inflammatory response characterized by a modest infiltration of granulocytes and macrophages whose numbers, after Day 7 postimplantation, remained unchanged for 21 days of sponge residence in the animal. In contrast, when the sponge contained tumor cells, there was a continuous increase in host cell infiltration that paralleled the increase in tumor cells. Whether in a sponge or not, tumor cells represented more than half of the recovered cells by Day 21 after tumor cell inoculation. Macrophages comprised the greatest fraction of host cells infiltrating the tumors. Flow cytometric analysis and morphological examination of disaggregated tumors indicated that macrophages (19-51%) made up the largest proportion of host cells followed in order by granulocytes (6-18%) and lymphocytes (2-9%). Sorting of marker-labeled cells revealed that 94% of the surface immunoglobulin-bearing cells were macrophages. Twenty-two % of the cells bearing the Thy 1.2 marker were lymphocytes, and 68% were macrophages. The data confirm the occurrence of a cellular host response in the tumor-bearing animal which is distinct from the foreign body reaction elicited by a blank sponge. Additionally, the sponge system described here represents a recoverable environment that would facilitate the study of in vivo host-tumor cell interactions that occur during early tumor development or later during therapy-induced tumor rejection when a palpable tumor is not present.
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Detailed studies on the application of three fluorescent antibiotics for DNA staining in flow cytometry.
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