Separation of Cells From a Murine Fibrosarcoma on the Basis of Size. I. Relationship Between Cell Size and Age as Modified by Growth In Vivo or In Vitro23

Grdina, David J.; Peters, Lester J.; Jones, Sandra L.; Chan, Edwin

doi:10.1093/jnci/61.1.209

Cited by 26 publications

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“…Separation by centrifugal elutriation.-FSa cells derived from pulmonary nodules were separated under sterile conditions by centrifugal elutriation using the same procedure described for their separation from either solid tumours or tissue culture (Grdina et al, 1978a(Grdina et al, , 1979. With the rotor speed set at 1525 rev/min, 2-3 x 108 cells, suspended in 20 ml of medium, were introduced into the elutriator chamber at a flow rate of 5 4 ml/min.…”

Section: Methodsmentioning

confidence: 99%

“…Cells collected in each fraction were counted by haemacytometer and by Coulter Counter (model ZB1; Coulter Electronics, Hialeah, FL), and their volume distributions determined with a multichannel analyser (Channelyzer II; Coulter Electronics). The modal volume was designated as that corresponding to the modal channel number of the volume distribution of each sample (Grdina et al, 1978a). F 1 containing small cells and cellular debris, and F 11 and 12, containing a heterogeneous mixture of cells, were discarded.…”

Section: Methodsmentioning

confidence: 99%

“…The DNA content of individual cells in suspension was determined by FMF using an ICP 11 flow cytometer (Phywe Co., Gottingen, Germany). cells were fixed in 70% ethanol and then stained with 50 mg/ml mithramycin (Mithracin; Pfizer and Co., Inc., New York, NY) in solution with MgCl2 (7 5 mM) and 12.5% aqueous ethanol (Grdina et al, 1978a). The resultant histograms of DNA fluorescence were computer-analysed (Johnston et al, 1978).…”

Section: Methodsmentioning

confidence: 99%

“…Because cell suspensions were derived directly from FSa lung nodules, a considerable number of normal diploid lung cells were present in all the elutriator fractions. Since FSa cells are heteroploid (i.e., 60-70 chromosomes) and contain 1-8 as much DNA as normal diploid cells , an estimate of the normal cell contamination in each of the tumourcell suspensions can be made by determining the area under the G1 normal diploid peak and dividing it by the area under the total FMF histogram (i.e., the area under both the tumour and normal peaks) (Grdina et al, 1978a). While the relative proportions of 4).…”

Section: Methodsmentioning

confidence: 99%

“…A limitation of this procedure, however, was the need for the inherent heterogeneity of the tumour-cell populations derived from solid tumours to be reduced for cell separation by centrifugal elutriation to be effective (Anderson et al, 1969;Grdina et al, 1978b). This was accomplished by a 48h in vitro incubation (Grdina et al, 1978a). To avoid this limitation, the method of centrifugal elutriation has been applied to separate murine fibrosarcoma (FSa) cells from pulmonary tumour nodules grown in C3H mice.…”

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confidence: 99%

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Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma pulmonary nodules

Grdina¹

1982

Br J Cancer

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Summary.-The cytotoxic effects in vivo of hydroxyurea (HU) on murine fibrosarcoma (FSa) cells grown as pulmonary tumours were determined. Tumour cells from 13-day-old nodules were made into suspension and separated on the basis of cell size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations, as well as the degree of contamination by normal diploid cells in each of the tumour-cell populations. HU cytotoxicity was tested by administering both a single 1 mg/g i.p. dose into mice that had been injected i.v. 20 min earlier with known numbers of synchronized viable FSa cells, and i.p. doses of 1 mg/g each into mice bearing 13-day-old pulmonary nodules. In the latter experiments, animals were killed 1 h after the last dose, and the tumour nodules were excised and made into a single-cell suspension and elutriated. Known numbers of cells from each fraction were injected into recipient mice to determine survival. In both sets of experiments, cell killing by HU correlated with the percentage of S-phase cells. The treatment of 13-day-old pulmonary nodules with 3 doses of HU also depleted the (G2+M) phase tumour cells and increased the heterogeneity between tumour subpopulations, as determined by FMF analysis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma pulmonary nodules

Grdina¹

1982

Br J Cancer

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Density distributions of human squamous carcinoma cells: Influence of growth conditions, proliferative status and DNA content

Keng

Sutherland

1986

Intl Journal of Cancer

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Human squamous carcinoma cells (A431, CaSki) were grown in vitro as multicellular spheroids or as exponential and plateau-phase monolayer cultures. Single-cell suspensions were obtained by disaggregating the spheroids and monolayer cultures with trypsin-EDTA. Their buoyant density distributions were then compared by centrifuging the suspensions for 30 min at about 800gave and 4 degrees C in linear, continuous Percoll gradients. We found differences in buoyant density related to the growth condition (i.e. cells grown as spheroids or plateau-phase cultures were more dense than those from exponential cultures) and to the cell-line (i.e. CaSki cells were more dense than the A431 cells). Cells isolated from the inner layers of the spheroids appeared to be less dense than those from the outer layers. In addition, quiescent cells separated from the A431 spheroids by centrifugal elutriation, a technique based on cell size, were more dispersed in buoyant density than the corresponding proliferating cells recovered from the same spheroids. Flow cytometric analysis with mithramycin was performed to measure the DNA content of the cells. We found that the CaSki cells contained relatively more DNA than the A431 cells whereas, for the same cell line, growth as monolayer cultures or as multicellular spheroids did not influence their relative DNA content. We conclude that the growth conditions, the proliferative status of the cells and possibly their relative DNA content may influence the density distribution of these cells.

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Shedding of mitotic cells from the surface of multicell spheroids during growth

Landry

Freyer

Sutherland

1981

Journal Cellular Physiology

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During the growth of EMT6/Ro mammary tumor multicell spheroids, a large number of cells are shed into the suspension medium. The rate of cell shedding was 218 cells per square millimeter of spheroid surface per hour, or up to 1.5% of the total spheroid cell content per hour. Shed cells had a clonogenic capacity equal to that of exponential monolayer cultures and were further characterized by volume distribution, mitotic index, flow cytofluorometry, and autoradiography. The results indicated that cells are released from the spheroid surface at mitosis, presumably due to a loosening of the cell-to-cell attachment during this cycle phase. These mitotic cells, when placed in monolayer culture, attached and grew synchronously with a cell cycle time of about 13 hours. Shed cells kept in suspension culture had a similar cell cycle time, but these cells reaggregated immediately after mitosis. The results indicated that cell shedding and reaggregation both occur near the time of mitosis and are intrinsic factors regulating the initiation and subsequent growth of multicell spheroids. Although these studies were done with spheroids cultured in vitro, shedding of mitotic cells may play an important role in the in vivo process of metastasis.

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Separation of Cells From a Murine Fibrosarcoma on the Basis of Size. I. Relationship Between Cell Size and Age as Modified by Growth In Vivo or In Vitro23

Cited by 26 publications

References 0 publications

Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma pulmonary nodules

Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma pulmonary nodules

Density distributions of human squamous carcinoma cells: Influence of growth conditions, proliferative status and DNA content

Shedding of mitotic cells from the surface of multicell spheroids during growth

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