An autoradiographic screening test for mycoplasmal contamination of mammalian cell cultures

Studzinski, George P.; Gierthy, John F.; Cholon, Jolanta J.

doi:10.1007/bf02615948

Cited by 92 publications

(23 citation statements)

References 14 publications

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“…Normal values for pyruvate carboxylase in leuk* cytes were 0.092 (0.070-0.208) mU/mg protein (n = 5), in lymphocytes 0.154 (0.092-0.262) mU/mg protein (n = 5), and in fibroblasts 1. 36 (0.778-2.19) mU/mg protein (n = 5). The patient with hepatic, renal, and cerebral pyruvate carboxylase deficiency had no detectable activity (<0.009 mU/mg protein) in his leukocytes and 0.018 mU/mg protein in his fibroblasts.…”

Section: Discussionmentioning

confidence: 99%

“…50 mM potassium phosphate buffer, pH 7.4, and 0.5 mM EDTA (Isohashi, Leiter, Utter medium: ILU medium) (20), and frozen at -76'. Cell scraping and culture medium were checked for bacterial and mycoplasmal contamination (36). Five to 6 million cells provided enough material to assay for pyruvate carboxylase, PEPCK, and citrate synthase.…”

Section: Fibroblast Culturementioning

confidence: 99%

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Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase Activity in Leukocytes and Fibroblasts from a Patient with Pyruvate Carboxylase Deficiency

1979

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Section: Discussionmentioning

confidence: 99%

Section: Fibroblast Culturementioning

confidence: 99%

Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase Activity in Leukocytes and Fibroblasts from a Patient with Pyruvate Carboxylase Deficiency

1979

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“…(FlLr, F1OLr) selected from these two sublines have been described in detail (13,18). All cells were grown in Eagle's minimal essential medium plus 10% fetal calf serum as described and were found to be uncontaminated by mycoplasma species by several assays (19)(20)(21)(22). Plasma membrane vesicles were harvested from the culture medium of actively growing roller bottle cultures of the various B16 sublines and purified as described (17).…”

Section: Introductionmentioning

confidence: 99%

Arrest and metastasis of blood-borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells.

Poste¹,

Nicolson²

1980

Proc. Natl. Acad. Sci. U.S.A.

226

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B16 mouse melanoma sublines in culture spontaneously shed intact plasma membrane vesicles. These vesicles can be fused with the plasma membrane of cells from homologous and heterologous B16 sublines by using polyethylene gycol and phytohemagglutinin-P. Fusion of vesicles from a highly metastatic subline (FlO) that localizes exclusively in the lung with cells from a poorly metastatic subline (Fl) The arrest of circulating tumor cells in the microcirculation is an essential step in the formation of hematogenous metastases (1-3). Little is presently known about the mechanisms involved in this process or the factors that influence blood-borne tumor cell arrest in different regions of the microcirculation. Clinical and experimental observations indicate that in many metastatic tumors the distribution and arrest patterns of circulating tumor cells are nonrandom (1-3), and malignant cells may localize preferentially in particular organs (4-11). A striking illustration of this phenomenon is provided by experiments showing that tumor cell lines that ordinarily metastasize to the lung also stop in pieces of lung, but not other organs, implanted at ectopic sites (9, 12; I. R. Hart, personal communication). The importance of tumor cell properties in determining cell arrest patterns in the microcirculation is also suggested by recent work in which a series of B16 mouse melanoma cell variant lines that metastasize preferentially to lung (13), liver (14), ovary (15), and brain (16) have been isolated from the same parent B16 line.In this paper we show that differences in the ability of B16 melanoma sublines to become arrested in the lung microcirculation are determined in part by the surface properties of these cells. By transferring plasma membrane components from a highly metastatic B16 subline (FlO) that localizes exclusively in the lung to another B16 subline (Fl), which ordinarily forms few metastases in the lung or other organs, we have been able to significantly increase the ability of F1 cells to become arrested in the lung and form pulmonary metastases. (FlLr, F1OLr) selected from these two sublines have been described in detail (13,18). All cells were grown in Eagle's minimal essential medium plus 10% fetal calf serum as described and were found to be uncontaminated by mycoplasma species by several assays (19)(20)(21)(22). Plasma membrane vesicles were harvested from the culture medium of actively growing roller bottle cultures of the various B16 sublines and purified as described (17). In certain experiments, vesicle surface proteins were labeled with 1251 or fluorescein isothiocyanate by using methods described previously (23, 24). Protein content was measured by the method of Lowry et al. (25).Vesicle-Cell Interactions. Purified vesicle preparations containing 30040 g of protein were incubated in suspension with 1 X 107 melanoma cells in minimal essential medium containing phytohemagglutinin-P (PHA; Difco) at 50 ,g/ml and 40% (vol/vol) polyethylene glycol, molecular weight 6000 (PEG) (PHA/PEG) for 5 mi...

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“…Cultures were incubated at 37 "C in an atmosphere of 10% C02 in air and a relative humidity of 80%. The cells were free of mycoplasma contamination as judged by absence of cytoplasmatic incorporation of 3H-labeled thymidine [20]. All experiments were carried out in culture media containing 10% calf serum.…”

mentioning

confidence: 99%

Prostaglandin E₂ Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

Claesson

Lindgren

Hammarström

1977

European Journal of Biochemistry

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High cellular adenosine 3' : 5 '-monophosphate (cyclic AMP) levels were found in a polyoma-virustransformed 3T3 fibroblast line, which produces comparatively high prostaglandin E2 concentrations. Prostaglandin E2 and cyclic AMP increased with time during 6 days of growth. Both effects were prevented by three different cyclo-oxygenase inhibitors. Addition of prostaglandin Ez, at a concentration which would have been synthesized in the absence of inhibitor, reversed the effect of indomethacin, one of the cyclo-oxygenase inhibitors, on cyclic AMP. It is concluded that endogenous prostaglandin E2 production in these transformed cells influences their cellular cyclic AMP levels.E-type prostaglandins can raise cyclic AMP levels in many tissues and cultured cells by stimulating adenylate cyclase [l]. A number of reports indicate that animal cells in culture produce prostaglandins (see e.g. [2-11 3). However, it has not been clear if this prostaglandin production has effects on cellular cyclic AMP levels since the amounts of prostaglandins produced are often orders of magnitude lower than the amounts added to raise cyclic AMP. The question if endogenous prostaglandin production can influence cyclic AMP levels in cell cultures is an important one since many reports indicate that cyclic AMP has essential roles in the regulation of cell proliferation and differentiation . The present report shows that prostaglandin E2 production by polyoma-virustransformed 3T3 cells raises their cellular cyclic AMP concentrations. MATERIALS AND METHODSDulbecco's modified Eagle's medium, calf serum, trypsin solution (2.5 %), and penicillin/streptomycin solution (10000 I.U./ml-10 mg/ml) were pur- Cell CulturesA polyoma-virus-transformed Balb/c 3T3 mouse embryo fibroblast line, originally cloned by Dr G. Todaro, was kindly given to us by Dr M. M. Burger (University of Basel, Switzerland). This cell line will be referred to as py 3T3. The cells were passaged with 0.25 % trypsin and planted at 2 x lo5 cells per 90-mm petri dish in 10 ml of Dulbecco's modified Eagle's medium containing 10% calf serum, 100 I.U./ ml of penicillin G and 0.1 mg/ml of streptomycin. Cultures were incubated at 37 "C in an atmosphere of 10% C02 in air and a relative humidity of 80%. The cells were free of mycoplasma contamination as judged by absence of cytoplasmatic incorporation of 3H-labeled thymidine [20]. All experiments were carried out in culture media containing 10% calf serum.

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An autoradiographic screening test for mycoplasmal contamination of mammalian cell cultures

Cited by 92 publications

References 14 publications

Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase Activity in Leukocytes and Fibroblasts from a Patient with Pyruvate Carboxylase Deficiency

Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase Activity in Leukocytes and Fibroblasts from a Patient with Pyruvate Carboxylase Deficiency

Arrest and metastasis of blood-borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells.

Prostaglandin E₂ Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

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An autoradiographic screening test for mycoplasmal contamination of mammalian cell cultures

Cited by 92 publications

References 14 publications

Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase Activity in Leukocytes and Fibroblasts from a Patient with Pyruvate Carboxylase Deficiency

Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase Activity in Leukocytes and Fibroblasts from a Patient with Pyruvate Carboxylase Deficiency

Arrest and metastasis of blood-borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells.

Prostaglandin E2 Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

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Prostaglandin E₂ Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels