An automated method for autoradiographic analysis of cultured Schwann cells

Baichwal, R. R.; Yan, Lu; Bosler, A.; DeVries, George H.

doi:10.1016/0165-0270(87)90062-8

Cited by 8 publications

(4 citation statements)

References 8 publications

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“…After incubation, cells were washed and processed for autoradiography as described (12). A minimum of 800-1000 cells per coverslip was counted, and a labeling index was determined by a semiautomated image analysis system (13).…”

Section: Methodsmentioning

confidence: 99%

Macrophage-mediated myelin-related mitogenic factor for cultured Schwann cells.

Baichwal

Bigbee

DeVries

1988

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

131

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Conditioned medium from cultured peritoneal macrophages that have phagocytosed a myelin membrane fraction is mitogenic for cultured Schwann cells. Production of the mitogenic supernatant was time-and dose-dependent with a maximal Schwann cell-proliferative response from supernatants after 48-hr incubation of cultured macrophages with myelin-enriched fraction (200 ,ug of protein per ml). The response was specific for myelin membrane: supernatants derived from macrophages incubated with axolemma, liver microsomes, polystyrene beads, or lipopolysaccharide were not mitogenic. Lysosomal processing of the myelin membrane was necessary for the production of the mitogenic factor, which was shown to be heat labile and trypsin sensitive. There was no species specificity because myelin membranes isolated from the central and peripheral nervous systems of rat, bovine, and human were equally potent in eliciting mitogenic supernatant. However, supernatants derived from central nervous system myelin membranes were two to three times more mitogenic than those obtained from peripheral nervous system fractions of the same species. Previous observations that myelin is mitogenic for cultured Schwann cells may, in part, involve the intermediate processing of myelin by macrophages that are present in Schwann cell cultures. These results suggest that macrophages play a crucial role in Schwann cell proliferation during Wallerian degeneration.The source of the mitogenic signal for Schwann cell division during Wallerian degeneration is not clear. Removal of myelin sheaths accompanying Wallerian degeneration has been ascribed to Schwann cells (1, 2) as well as macrophages (3-5). Beuche and Friede (5) observed that Wallerian degeneration proceeding without nonresident phagocytic cells (macrophages) showed no Schwann cell proliferation and no active intracellular digestion of myelin-implying that myelin membranes were removed solely by macrophages after nerve degeneration. This observation also suggests that Schwann cell proliferation is related to myelin removal by macrophages. Salzer and Bunge (6) using dorsal root ganglion explant cultures showed that only myelin-related Schwann cells proliferate after axotomy. In contrast, however, in vivo studies by Pellegrino et al. (7) (9). The presence of macrophages in our Schwann cell cultures and the possible involvement of macrophages in removal of myelin debris during Wallerian degeneration (10) prompted us to study the role of macrophages in mediating mitogenicity of myelin for cultured Schwann cells. Using rat peritoneal macrophages, we have shown that macrophages stimulated with MEF produce a soluble factor(s) mitogenic for cultured Schwann cells. Production of the soluble mitogenic factor shows a time-and dose-dependent response. Other membrane fractions or nonspecific agents do not stimulate macrophages to produce a mitogenic-conditioned medium. The myelin membrane undergoes lysosomal processing in the macrophage before mitogenic factor is produced. Sensitivity of the mitogenic sup...

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Section: Methodsmentioning

confidence: 99%

Macrophage-mediated myelin-related mitogenic factor for cultured Schwann cells.

Baichwal

Bigbee

DeVries

1988

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

131

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“…It thus decreased the processing time. Meanwhile, it allowed to get entire cell contours instead of only cell body contours (Clements and Buzy, 1991) or nuclei's contours (Baichwal et al, 1987). The performance of this cell detection procedure was evaluated.…”

Section: Discussionmentioning

confidence: 99%

“…There is, however, a scarcity of reports for unstained cell study because of the difficulties encountered in detecting such cells. Techniques like the recognition of nuclei (Baichwal et al, 1987) and the determination of cell size range (Clements and Buzy, 1991) have been used in cell counting. These methods do not provide yet precise morphological information for individual cells.…”

Section: Introductionmentioning

confidence: 99%

Automated image analyzing system for the quantitative study of living cells in culture

Opstal

Ranger²,

Lejeune³

et al. 1994

Microscopy Res & Technique

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A fully automated image analyzing system was developed for the quantitative study of cells in culture. It was able to count cells, to classify cells according to their morphological characteristics and to follow cell culture development. A specific procedure was designed to process Hoffman modulation contrast images. It detects local gray level differences while using conditional dilation techniques. We were able to successfully detect aggregated unstained cells, presently a technical limit in image segmentation. Living cells can be studied in a noninvasive and nondestructive way with this system. An improved automatic focusing algorithm was developed which ensured an accurate prediction of the optimal focus position. A strictly defined sampling procedure was applied to estimate unbiasedly cell density and obtain precisely cell contours. The evaluation of the system was carried out on Chinese hamster ovary (CHO-NTR) cell cultures treated with a newly developed neurotensin agonist JMV449. Chinese hamster ovary cell division was found to be retarded 20 hours after the JMV449 treatment, while the morphology of CHO-NTR cells has already undergone significant changes 12 hours after the treatment. This image analyzing system provides the possibility to follow cell culture development (e.g., cell density evolution, cell morphological changes) under various experimental conditions.

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“…Twentyfour hours later, 1.5 pCi of [3H]thymidine in 125 pl of 10% DME was added to each well. Two days later, the cells were processed for autoradiography (DeVries et al, 1983~;Baichwal et al, 1987). A minimum of 1,000 cells was counted per coverslip to determine the percentage of cells which had accumulated [3H]thymidine.…”

Section: Autoradiography Of Schwann Cell Culturesmentioning

confidence: 99%

1‐Oleoyl‐2‐Acetylglycerol and A23187 Potentiate Axolemma‐and Myelin‐Induced Schwann Cell Proliferation

Saunders

DeVries

1988

Journal of Neurochemistry

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The effects of 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore A23187 on the proliferation of Schwann cells stimulated with either a myelin-enriched membrane fraction (MEF) or an axolemma-enriched membrane fraction (AEF) have been examined. Using incorporation of [3H]thymidine as an index of proliferation, 16% of the cells became labeled after incubation with MEF (20 micrograms protein/ml) and AEF (40 micrograms protein/ml) for 72 h. Only 0.5% of the cells became labeled in cultures which were not exposed to the membrane fractions. Addition of OAG (10-500 microM) or A23187 (1.9-190 nM) in the absence of the membrane mitogens had no effect on the proliferative response of quiescent cultures of Schwann cells. When added simultaneously, however, OAG and A23187 were able to induce proliferation of the cells, although the response was only 30% of the response achieved with maximal doses of either AEF or MEF. Both OAG and A23187 were able to potentiate the mitogenicity of AEF or MEF, but only when AEF and MEF were added at submaximal concentrations. When Schwann cells were prelabeled with [3H]glycerol and then stimulated to proliferate with AEF or MEF, the amount of [3H]diacylglycerol was increased two- to threefold above that in control cultures for time periods up to 1 h. These results suggest that the proliferation of Schwann cells induced by either AEF or MEF is partially mediated through the combined effects of diacylglycerol and an increase in intracellular calcium.

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An automated method for autoradiographic analysis of cultured Schwann cells

Cited by 8 publications

References 8 publications

Macrophage-mediated myelin-related mitogenic factor for cultured Schwann cells.

Macrophage-mediated myelin-related mitogenic factor for cultured Schwann cells.

Automated image analyzing system for the quantitative study of living cells in culture

1‐Oleoyl‐2‐Acetylglycerol and A23187 Potentiate Axolemma‐and Myelin‐Induced Schwann Cell Proliferation

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