scholarly journals Cell membrane rupture: A novel test reveals significant variations among different brands of tissue culture flasks

2020 ◽  
Author(s):  
Ruy Tchao
Keyword(s):  
Tissue Culture ◽  
Cell Membrane ◽  
Surface Heterogeneity ◽  
Assay Method ◽  
Simple Test ◽  
Large Variability ◽  
Fluorescent Marker ◽  
Polystyrene Surface ◽  
Membrane Rupture ◽  
Marker Proteins

Abstract Objectives Loss of cytoplasmic molecules including protein controls, due to cell membrane rupture can cause errors and irreproducibility in research data. Previous results have shown that during the washing of a monolayer of cells with a balanced salt solution, the fluid force causes cell membrane rupture on some areas of the flasks/dishes. This fact shows the non-uniformity of the polystyrene surface in terms of cell culture. There is at present no simple test to monitor that surface. This paper presents a novel biologically based assay to determine the degree of heterogeneity of flasks supplied by various manufacturers. Results This paper shows that significant variation exists in polystyrene surface heterogeneity among several brands of tissue culture flasks, varying from 4% to 20% of the flask surface. There is also large variability within the production lot of a manufacturer. The assay method involves loading the cells with a cytoplasmic fluorescent marker that is released upon cell membrane rupture. Cell membrane rupture also causes the loss of marker proteins such as GAPDH used in Westernblots. This novel assay method can be used to monitor the batch consistency and the manufacturing process of flasks/dishes. It may also be used to test new biomaterials.

scholarly journals Cell membrane rupture: a novel test reveals significant variations among different brands of tissue culture flasks

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Ruy Tchao
Keyword(s):  
Tissue Culture ◽  
Cell Membrane ◽  
Surface Heterogeneity ◽  
Assay Method ◽  
Simple Test ◽  
Large Variability ◽  
Fluorescent Marker ◽  
Polystyrene Surface ◽  
Membrane Rupture ◽  
Marker Proteins

Abstract Objectives Loss of cytoplasmic molecules including protein controls, due to cell membrane rupture can cause errors and irreproducibility in research data. Previous results have shown that during the washing of a monolayer of cells with a balanced salt solution, the fluid force causes cell membrane rupture on some areas of the flasks/dishes. This fact shows the non-uniformity of the polystyrene surface in terms of cell culture. There is at present no simple test to monitor that surface. This paper presents a novel biologically based assay to determine the degree of heterogeneity of flasks supplied by various manufacturers. Results This paper shows that significant variation exists in polystyrene surface heterogeneity among several brands of tissue culture flasks, varying from 4 to 20% of the flask surface. There is also large variability within the production lot of a manufacturer. The assay method involves loading the cells with a cytoplasmic fluorescent marker that is released upon cell membrane rupture. Cell membrane rupture also causes the loss of marker proteins such as GAPDH used in Westernblots. This novel assay method can be used to monitor the batch consistency and the manufacturing process of flasks/dishes. It may also be used to test new biomaterials.

scholarly journals Cell Membrane Rupture: A Novel Test Reveals Significant Variations Among Different Brands of Tissue Culture Flasks

2020 ◽  
Author(s):  
Ruy Tchao
Keyword(s):  
Tissue Culture ◽  
Cell Membrane ◽  
Assay Method ◽  
Adherent Cells ◽  
Fluid Shear ◽  
Large Variability ◽  
Fluorescent Marker ◽  
Polystyrene Surface ◽  
Membrane Rupture ◽  
Marker Proteins

Unanticipated errors in scientific research data can be attributed to the unwarranted assumption of uniformity in the polystyrene surface that is ubiquitously used in tissue culture flasks and dishes. We have shown that when adherent cells are subjected to fluid shear force, equivalent to rinsing the culture with a balanced salt solution, cells on some areas of the polystyrene surface will immediately rupture while still adherent on the surface. This heterogeneity on the polystyrene surface can cause unexpected variability in experimental results and in replicating experiments among labs. In this paper a novel quantitative method is described to measure the degree of heterogeneity on the polystyrene surface of tissue culture flasks. The results show significant variation among several brands of tissue culture flasks as well as large variability within the production lot of a manufacturer. The assay method involves loading the cells with a fluorescent marker that is released upon membrane rupture. Cell membrane rupture also causes the loss of marker proteins used in Westernblots. This novel assay method can be used to monitor the batch consistency and the manufacturing process of flasks and dishes. It may also be used to test new biomaterials.

Analysis of Lysophagic Flux in Cultured Cells using Lyso-Keima v1

2021 ◽  
Author(s):  
Vinay V. Eapen ◽  
Sharan Swarup ◽  
Melisa Hoyer ◽  
Harper not provided JW
Keyword(s):  
Tissue Culture ◽  
Cultured Cells ◽  
Confocal Imaging ◽  
Signaling Proteins ◽  
Membrane Rupture ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Luminal Side ◽  
Galectin 3 ◽  
Autophagy Pathway

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a facile method for monitoring lysophagy using the acid sensitive fluorophore mKeima, affixed onto Galectin-3, which allows for the monitoring of lysophagic flux by Flow cytometry, Western blotting or Confocal imaging. This method, which we have termed Lyso-Keima, serves as a facile and quantitative assay for monitoring lysophagy in tissue culture cells.

Analysis of Lysophagic Flux in Cultured Cells using Lyso-Keima v2

2021 ◽  
Author(s):  
Vinay V. Eapen ◽  
Sharan Swarup ◽  
Melisa Hoyer ◽  
Harper JW
Keyword(s):  
Tissue Culture ◽  
Cultured Cells ◽  
Confocal Imaging ◽  
Signaling Proteins ◽  
Membrane Rupture ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Luminal Side ◽  
Galectin 3 ◽  
Autophagy Pathway

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a facile method for monitoring lysophagy using the acid sensitive fluorophore mKeima, affixed onto Galectin-3, which allows for the monitoring of lysophagic flux by Flow cytometry, Western blotting or Confocal imaging. This method, which we have termed Lyso-Keima, serves as a facile and quantitative assay for monitoring lysophagy in tissue culture cells.

Role of cell membrane rupture in the pathogenesis of electrical trauma

1988 ◽  
Vol 44 (6) ◽  
pp. 709-719 ◽  
Author(s):  
Raphael C. Lee ◽  
Diane C. Gaylor ◽  
Deepak Bhatt ◽  
David A. Israel
Keyword(s):  
Cell Membrane ◽  
Membrane Rupture

scholarly journals THE TOXICITY OF STREPTOLYSIN O FOR BEATING MAMMALIAN HEART CELLS IN TISSUE CULTURE

1970 ◽  
Vol 131 (4) ◽  
pp. 745-763 ◽  
Author(s):  
A. Thompson ◽  
S. P. Halbert ◽  
U. Smith
Keyword(s):  
Tissue Culture ◽  
Cell Membrane ◽  
Culture Fluid ◽  
Ultrastructural Level ◽  
Cardiac Cells ◽  
Heart Cells ◽  
Streptolysin O ◽  
Multiple Cell ◽  
Bleb Formation

Pulsating mammalian myocardial cells were found to be highly susceptible in tissue culture to rapid destruction by streptolysin O. Cessation of beating occurred almost immediately, followed within minutes by multiple cell membrane bleb formation. Parallel with these changes, the cytoplasm became intensely granular and the nuclear membrane apparently thickened when viewed by phase microscopy. At the ultrastructural level, the cell membrane blebs were found to contain relatively small numbers of granular fragments. The endoplasmic reticulum of damaged heart cells was quite swollen, and its contents were considerably condensed. The myofibers were not strikingly altered, but cytoplasmic and mitochondria vacuoles were rather abundant. Cardiac endothelial, kidney epithelial, and fibroblast cells were also susceptible to lysis by this toxin, but the reactions occurred more slowly or bleb formation was less evident. An antiserotonin drug known to be protective against streptolysin-O in vivo (UML-491), did not protect against killing of cardiac cells at the tissue culture level. Serotonin could not be detected in the culture fluid after lysis of cardiac cells by streptolysin O.

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