PubMed Health⌕ Search

Biomedical subjects

T K Sharpless

Publications and source records attributed to T K Sharpless.

9 recordsLinked to original sources
PubMed1979

Correlation between cell cycle duration and RNA content.

The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively. Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intercellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content. The data suggest that the rate of progression through the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.

Animals↗
PubMed1979

Cell-cycle distribution of urothelial tumour cells as measured by flow cytometry.

The fraction of cells in S + G2 + mitosis from 54 urothelial tumours was calculated by flow cytometry after acridine orange (AO) staining of cells obtained by bladder irrigation or biopsy. Fluorescence signals emitted by the AO-stained DNA and RNA of each cell were separated optically and measured for 5,000 cells per specimen. The patients were classified by the histology of their tumours and clinical data into 5 diagnostic categories: NED (no evidence of disease, but history of bladder tumour), 3; papilloma, 8; non-invasive papillary carcinoma, 8; carcinoma in situ, 17 and invasive carcinoma, 18. The fraction of cells with DNA values in S + G2 + M of the cell cycle varied between 7 and 57% of the total, with a wide range within each diagnostic category, but no statistically significant differences between the groups. The proportion of cells in S + G2 + M from an individual tumour was not correlated with histologic grade or clinical behaviour. The possibility that some tumour cells with DNA values above G1 level are quiescent cells arrested at S or G2 is discussed.

Cell Count↗
PubMed1979

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.

Cytological Techniques↗
PubMed1979

Quantitation of lymphocyte response to PHA by flow cytofluorometry. III. Heterogeneity of induction period.

Early events in phytohaemagglutinin (PHA) stimulation of mouse splenocytes have been quantitated by using flow cytometry and supravital staining with acridine orange (AO). Increasing percentages of single cells with increased metachromatic (red) AO staining were demonstrated in cultures stimulated by PHA for up to 24 hr. These differences in staining could be eliminated by fixation with 1:1 ethanol/acetone before staining. Stimulated cells showed an increase in nonspecific esterase activity as measured by flow cytometry after supravital staining with fluorescein diacetate (FDA). The data reported show a heterogeneity in the per cell response of mouse splenocytes to PHA. The relationship between these data and the mechanism of mitogen stimulation is discussed.

Acridine Orange↗
PubMed1979

Regional lymph node reactivity in explanted bladder cancer of mice as measured by flow cytometry.

The reactivity of lymphocytes in lymph nodes draining the site of a transplantable experimental bladder tumor (MBT2 in C3H/HeJ mice) has been measured in a multiparameter flow cytometry system. Acridine orange was used as a nucleic acid probe. This dye intercalates in helical DNA, emitting green (530 nm) fluorescence upon exposure to blue light; it stacks to single-stranded RNA, emitting red (640 nm) fluorescence. The relative magnitude of the increase of lymphocyte DNA and RNA has been evaluated simultaneously in tumor-draining nodes, in nondraining nodes of the same animal, and in untreated control animals. Stimulation of the regional node lymphocytes could be observed after 20 days but not after 10 days. It was uniformly high at 35 days. The transcriptive response (increased proportion of lymphocytes with high RNA) was more pronounced than the proliferative (increased proportion of lymphocytes with more than diploid DNA). The histological changes in the stimulated nodes resembled closely those described by others in human tumor-draining nodes. The described method has the advantage of being simple, rapid, and able to measure a representative part of the whole-cell population.

Animals↗
PubMed1977

Cell cycle-related changes in nuclear chromatin of stimulated lymphocytes as measured by flow cytometry.

Flow cytometric techniques have been developed to assay lymphocyte stimulation as reflected by the increase in the cell transcriptional activity and cell progression through the cell cycle. The metachromatic fluorescent dye, acridine orange, is used to (a) stain DNA and RNA differentially in individual cells, and (b) stain nuclear chromatin after removal of cellular RNA BY RNase and cell pretreatment at acidic pH. Stimulated cells with diploid DNA content (G1) have an increased content of stainable RNA that makes it possible to distinguish them from nonstimulated (G0) cells. G0 cells can also be distinguished from G1 cells based on differences in stainability of their nuclear chromatin after treatment with acid. Mitotic indices can be scored automatically, inasmuch as the metaphase chromatin stains differently than does chromatin in the interphase cells. Altogether, the numbers of cells in the G0, G1, S, G2, and M phases may be obtained rapidly and with great accuracy. The cell transciptional activity can be correlated with changes in nuclear chromatin (e.g., during the transition from G0 to G1). The two independent techniques may also prove to be useful in recognizing and quantitating noncycling cells in other cell systems. The possible mechanisms responsible for differential stainability of nuclear chromatin in cells at different phases of the cell cycle are discussed.

Cell Cycle↗