Method for the quantitative evaluation of data from flow microfluorometry

Fried, Jerrold

doi:10.1016/0010-4809(76)90006-9

Cited by 179 publications

(56 citation statements)

References 6 publications

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“…Attempts were thus made to work with cells within a defined range of passage numbers, and parallel controls were run in culture medium identical to that utilized for 19.0 f 0.7 (8) 21.0 f 1.2 (8) 13.9 f 1.7 (6) 13. Table 6 Effect Fried (14). (20).…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

et al. 1982

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DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities, and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 pg/ml MMC, 3HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of 3HdT incorporation than did those sorted from the first half of S.Fanconi's anemia cells exhibited a marked accumulation in the Gz + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with 0.05 pg/ ml MMC, the G2 + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.2. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a Gz + M increment than did FA cells, even after exposure to 0.1 pg/ml MMC. Examination of cells sorted from the Gz + M peak revealed that MMC-treated FA cells were blocked prior to mitosis.To determine whether the response of FA cells was specific for a bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G2 + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.Key terms: Fanconi's anemia, DNA crosslinking agent, mitomycin C, flow cytometry, 33342 Hoechst, Gz block, Cell cycle analysis, DNA repair Fanconi's anemia (FA), a syndrome of pancytopenia and a variable constellation of congenital anomalies (12,34,44), has been shown to be associated with chromosomal instability (8, 42, 47) and a predisposition for cancer (5,16, 43). Cells from patients with FA show increased sensitivity to agents capable of producing DNA crosslinks. This sensitivity is evident as decreased survival (51), increased chromosome breakage (3, 29,40) and a reduction in both the rate of progression through I This research was supported by grants CD-36F from the American Cancer Society and GM21121 from the National Institute of General Medical Sciences.Presented at the VIII Conference on Analytical Cytology and Cytometry, Wentworth-by-the-Sea, New Hampshire, May 19-25, 1981. the cell cycle (41) and in the proportion of mitotic cells (17, 51), following exposure to such agents.A reduction in the ability of cells from patients with Fanconi's anemia to remove DNA crosslinks, caused e.g. by mitomycin C (MMC), was observed in some studies (15) but not in others (13, 25). There are also isolated reports that cells from patients with FA exhibit other defects, such as a reduced ability to remove damage induc...

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Section: Discussionmentioning

confidence: 99%

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

et al. 1982

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“…Cells were collected in flasks containing six slide strips (made by cutting microscope slides in half lengthwise) and 150 pCi (per flask) of tritiated thymidine (NEN, 50 Ci/mmole). Slide strips were removed at 5,6,7,8,9,10,11,12 and 24 hr postselection, washed four times in 50 ml of Eagle's medium (at 37°C) containing M cold thymidine, four times in 50 ml of cold PBS, fixed in -20°C absolute ethanol, air dried and stored at 4°C. The percentage labeled cells was determined by microscopic observation of at least 500 cells at a magnification of ~5 0 0 .…”

Section: Methodsmentioning

confidence: 99%

Characterization of the cell cycle and synchrony in a CV‐1 cell line

D'Alisa¹,

Gershey²

1980

Cytometry

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Synchronous populations of CV-1 cells (17) provides a means for obtaining relatively large populations of synchronous cells with a minimal amount of perturbation to the cell cycle, we have found that a dispersion in the synchrony exists, though smaller than that produced by other methods for synchronizing mammalian cells. Using standard techniques, we have developed an approach to evaluating the degree of synchrony and identifying causes for its imperfection. This approach and our findings should be applicable to analysis of various cell lines and synchrony systems.The degree of synchrony in subcloned cells of the CV-1 line was monitored at times of selection, attachment, S phase entry and the following cell division. Although populations of cells in GI, early, middle and late S, GP and M phases of the cell cycle were clearly resolved, 90% of the cells were dispersed within a 3-hr period throughout an average cell cycle length of 19 hr. Several factors besides cell age contribute to the variability in the length of the GI phase and the imperfect synchrony achieved by the automated selection of mitotic CV-1 cells. During the first cycle after selection, a major factor appears to be interruption of anchorage dependent growth following removal of the mitotic cells from roller cultures.

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“…Since our samples came from a partially synchronized population, the parametric method proposed by Fried (1976) was applied to deconvolve histograms into phase fractions (Dean 1985). A multiple linear regression program in the SPSS statistical package (SPSS Inc.) was used to obtain the relative area under each normai curve by the ieast-square fitting technique (Fried 1976). A series of 14 normal curves were utilized at the beginning of the regression.…”

Section: Micro-micromentioning

confidence: 99%

Species-specific phytoplankton growth rates via diel DNA synthesis cycles. II DNA quantification and model verification in the dino-flagellate Heterocapsa triquetra

Chang¹,

Carpenter²

1988

Mar. Ecol. Prog. Ser.

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The DNA content of individual cells, after being stained by a DNA-specific fluorescent dye, was quantified by a TV camera-based microfluorometry system. Under proper dye concentrations, fluorescence intensity of a stained nucleus was in proportion to the amount of nuclear DNA. Durations of die1 cell cycle phases were estimated with DNA histograms from 2 partially synchronized cultures of the dinoflagellate Heterocapsa tn'quetra. Estimates of growth rate were calculated from phase durations and compared to the growth rates estimated by cell counts. Growth rates calculated by various mathemahcal procedures and sampling intervals were simllar, and the calculated growth rate via DNA synthesis gave a reasonably good estimate of the rate calculated by increase in cell density. This technique is promising in its application to species-specific growth rates in the field.

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Method for the quantitative evaluation of data from flow microfluorometry

Cited by 179 publications

References 6 publications

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

Characterization of the cell cycle and synchrony in a CV‐1 cell line

Species-specific phytoplankton growth rates via diel DNA synthesis cycles. II DNA quantification and model verification in the dino-flagellate Heterocapsa triquetra

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