Processing Immunoperoxidase Labeled Cell Monolayers and Cryostat Sesctions For Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 714-715
Author(s):  
K. Chien ◽  
R. Van de Velde ◽  
I.P. Shintaku ◽  
A.F. Sassoon
Keyword(s):  
Cell Monolayer ◽  
Cell Types ◽  
Surface Antigens ◽  
Immunoperoxidase Method ◽  
Reaction Step ◽  
Hot Plate ◽  
Air Drying ◽  
New Approach ◽  
Cell Monolayers ◽  
Glass Slides

Immunoelectron microscopy of neoplastic lymphoma cells is valuable for precise localization of surface antigens and identification of cell types. We have developed a new approach in which the immunohistochemical staining can be evaluated prior to embedding for EM and desired area subsequently selected for ultrathin sectioning.A freshly prepared lymphoma cell suspension is spun onto polylysine hydrobromide- coated glass slides by cytocentrifugation and immediately fixed without air drying in polylysine paraformaldehyde (PLP) fixative. After rinsing in PBS, slides are stained by a 3-step immunoperoxidase method. Cell monolayer is then fixed in buffered 3% glutaraldehyde prior to DAB reaction. After the DAB reaction step, wet monolayers can be examined under LM for presence of brown reaction product and selected monolayers then processed by routine methods for EM and embedded with the Chien Re-embedding Mold. After the polymerization, the epoxy blocks are easily separated from the glass slides by heatingon a 100°C hot plate for 20 seconds.

A pre-embedding, protein a-gold immunolabelling technique for cytocentrifuged cell monolayers for lm and tem

1986 ◽  
Vol 44 ◽  
pp. 220-221
Author(s):  
K. Chien ◽  
I.P. Shintaku ◽  
A.F. Sassoon ◽  
R.L. Van de Velde ◽  
R. Heusser
Keyword(s):  
Cell Surface ◽  
Staining Method ◽  
Protein A ◽  
Surface Antigens ◽  
Cell Suspensions ◽  
Cellular Morphology ◽  
Cellular Phenotype ◽  
Cell Surface Antigens ◽  
Cell Monolayers ◽  
Diagnostic Histopathology

Identification of cellular phenotype by cell surface antigens in conjunction with ultrastructural analysis of cellular morphology can be a useful tool in the study of biologic processes as well as in diagnostic histopathology. In this abstract, we describe a simple pre-embedding, protein A-gold staining method which is designed for cell suspensions combining the handling convenience of slide-mounted cell monolayers and the ability to evaluate specimen staining specificity prior to EM embedding.

scholarly journals Energetics of mesoscale cell turbulence in two-dimensional monolayers

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shao-Zhen Lin ◽  
Wu-Yang Zhang ◽  
Dapeng Bi ◽  
Bo Li ◽  
Xi-Qiao Feng
Keyword(s):  
Kinetic Energy ◽  
Tumor Metastasis ◽  
Cell Types ◽  
Boltzmann Distribution ◽  
Scaling Exponent ◽  
Biological Processes ◽  
Two Dimensional ◽  
Cell Monolayers ◽  
Wide Range ◽  
Epithelial Cell Monolayers

AbstractInvestigation of energy mechanisms at the collective cell scale is a challenge for understanding various biological processes, such as embryonic development and tumor metastasis. Here we investigate the energetics of self-sustained mesoscale turbulence in confluent two-dimensional (2D) cell monolayers. We find that the kinetic energy and enstrophy of collective cell flows in both epithelial and non-epithelial cell monolayers collapse to a family of probability density functions, which follow the q-Gaussian distribution rather than the Maxwell–Boltzmann distribution. The enstrophy scales linearly with the kinetic energy as the monolayer matures. The energy spectra exhibit a power-decaying law at large wavenumbers, with a scaling exponent markedly different from that in the classical 2D Kolmogorov–Kraichnan turbulence. These energetic features are demonstrated to be common for all cell types on various substrates with a wide range of stiffness. This study provides unique clues to understand active natures of cell population and tissues.

Cell Monolayer Deformation Microscopy reveals mechanical fragility of cell monolayers following EMT

2022 ◽  
Author(s):  
Amy A. Sutton ◽  
Clayton W. Molter ◽  
Ali Amini ◽  
Johanan Idicula ◽  
Max Furman ◽  
...  
Keyword(s):  
Cell Monolayer ◽  
Cell Monolayers

Na-K-Cl cotransport regulates intracellular volume and monolayer permeability of trabecular meshwork cells

1995 ◽  
Vol 268 (4) ◽  
pp. C1067-C1074 ◽  
Author(s):  
M. E. O'Donnell ◽  
J. D. Brandt ◽  
F. R. Curry
Keyword(s):  
Trabecular Meshwork ◽  
Ethacrynic Acid ◽  
Vascular Endothelial Cells ◽  
Critical Role ◽  
Cell Types ◽  
Cell Monolayers ◽  
Aqueous Humor Outflow ◽  
Trabecular Meshwork Cells ◽  
Intracellular Volume ◽  
Underlying Mechanisms

The trabecular meshwork (TM) of the eye plays a critical role in modulating intraocular pressure (IOP) through regulation of aqueous humor outflow, although the underlying mechanisms remain unknown. Ethacrynic acid, an agent known to inhibit Na-K-Cl cotransport of a number of cell types, recently has been reported to increase aqueous outflow and lower IOP through an unknown effect on the TM. In vascular endothelial cells and a variety of other cell types, the Na-K-Cl cotransporter functions to regulate intracellular volume. The present study was conducted to evaluate TM cells for the presence of Na-K-Cl cotransport activity and to test the hypothesis that modulation of cotransport activity alters intracellular volume and, consequently, permeability of the TM. We demonstrate here that bovine and human TM cells exhibit robust Na-K-Cl cotransport activity that is inhibited by bumetanide and by ethacrynic acid. Our studies also show that TM cell Na-K-Cl cotransport is modulated by a variety of hormones and neurotransmitters. Inhibition of the cotransporter either by bumetanide, ethacrynic acid, or inhibitory hormones reduces TM intracellular volume, whereas stimulatory hormones increase cell volume. In addition, shrinkage of the cells by hypertonic media stimulates cotransport activity and initiates a subsequent regulatory volume increase. Permeability of TM cell monolayers, assessed as transmonolayer flux of [14C]sucrose, is increased by hypertonicity-induced cell shrinkage and by bumetanide. These findings suggest that Na-K-Cl cotransport of TM cells is of central importance to regulation of intracellular volume and TM permeability. Defects of Na-K-Cl cotransport may underlie the pathophysiology of glaucoma.

scholarly journals Transcriptional profiling predicts running promotes cerebrovascular remodeling in young but not midlife mice

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Kate E. Foley ◽  
Hongtian Stanley Yang ◽  
Leah C. Graham ◽  
Gareth R. Howell
Keyword(s):  
Cognitive Decline ◽  
Transcriptional Profiling ◽  
Cell Types ◽  
Brain Health ◽  
New Approach ◽  
Age Related ◽  
Voluntary Running ◽  
Differential Gene ◽  
Age Related Cognitive Decline ◽  
Potential Therapeutic Intervention

Abstract Background The incidence of dementia and cognitive decline is increasing with no therapy or cure. One of the reasons treatment remains elusive is because there are various pathologies that contribute to age-related cognitive decline. Specifically, with Alzheimer’s disease, targeting to reduce amyloid beta plaques and phosphorylated tau aggregates in clinical trials has not yielded results to slow symptomology, suggesting a new approach is needed. Interestingly, exercise has been proposed as a potential therapeutic intervention to improve brain health and reduce the risk for dementia, however the benefits throughout aging are not well understood. Results To better understand the effects of exercise, we preformed transcriptional profiling on young (1–2 months) and midlife (12 months) C57BL/6 J (B6) male mice after 12 weeks of voluntary running. Data was compared to age-matched sedentary controls. Interestingly, the midlife running group naturally broke into two cohorts based on distance ran - either running a lot and more intensely (high runners) or running less and less intensely (low runners). Midlife high runners had lower LDL cholesterol as well as lower adiposity (%fat) compared to sedentary, than midlife low runners compared to sedentary suggesting more intense running lowered systemic markers of risk for age-related diseases including dementias. Differential gene analysis of transcriptional profiles generated from the cortex and hippocampus showed thousands of differentially expressed (DE) genes when comparing young runners to sedentary controls. However, only a few hundred genes were DE comparing either midlife high runners or midlife low runners to midlife sedentary controls. This indicates that, in our study, the effects of running are reduced through aging. Gene set enrichment analyses identified enrichment of genes involved in extracellular matrix (ECM), vascular remodeling and angiogenesis in young runners but not midlife runners. These genes are known to be expressed in multiple vascular-related cell types including astrocytes, endothelial cells, pericytes and smooth muscle cells. Conclusions Taken together these results suggest running may best serve as a preventative measure to reduce risk for cerebrovascular decline. Ultimately, this work shows that exercise may be more effective to prevent dementia if introduced at younger ages.

scholarly journals Modulation of Insulin Gene Expression with CRISPR/Cas9-based Transcription Factors

2021 ◽  
Vol 9 (A) ◽  
pp. 876-881
Author(s):  
Bakhytzhan Alzhanuly ◽  
Zhussipbek Y. Mukhatayev ◽  
Dauren M. Botbayev ◽  
Yeldar Ashirbekov ◽  
Nurlybek D. Katkenov ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cell Types ◽  
Diabetic Patients ◽  
Type I ◽  
Transcription Activator ◽  
New Approach ◽  
Donor Cells ◽  
Diabetes Type I ◽  
Promising Solution ◽  
Insulin Gene Expression

Background: The discovery and use of CRISPR/Cas9 technology have enabled researchers throughout the globe to continuously edit genomes for the benefit of science and medicine. Diabetes type I is one field of medicine where CRISPR/Cas9 has a strong potential for cell therapy development. The long-lasting paucity of healthy cells for clinical transplantation into diabetic patients has led to the search of new methods for producing β-cells from other human cell types. Embryonic stem cells are being studied worldwide as one most promising solution of this need. Aim: The aim of the study is to to check the feasibility of modulating human insulin transcription using CRISPR/Cas9-based synthetic transcription regulation factors. Results: A new approach for creating potential therapeutic donor cells with enhanced and suppressed insulin production based on one of the latest achievements of human genome editing was developed. Both synthetic transcription activator (VP64) and transcription repressor (KRAB) proteins were shown to function adequately well as a part of the whole CRISPR/Cas9-based system. We claim that our results have a lot to offer and can bring light to many studies where numerous labs are struggling on to treat this disease.

Cell behaviour in a polygonal cell sheet*

Development ◽  
1984 ◽  
Vol 83 (Supplement) ◽  
pp. 313-327
Author(s):  
H. Honda ◽  
R. Kodama ◽  
T. Takeuchi ◽  
H. Yamanaka ◽  
K. Watanabe ◽  
...  
Keyword(s):  
Cell Sheet ◽  
Cell Monolayer ◽  
Time Lapse ◽  
Geometrical Method ◽  
Cell Behaviour ◽  
Cell Monolayers ◽  
Polygonal Cell ◽  
Computer Based ◽  
Insight Into

Cell monolayers on culture dishes were divided into two groups: tensile monolayers and non-tensile ones. In the development of an epithelium, a non-tensile cell monolayer turns into a tightly bound tensile one. Detection of these states was carried out by using the boundary shortening procedure, a computer-based geometrical method to show how much the polygonal cell boundary contracts. Non-tensile monolayers were divided further into two groups according to their motility: a fluctuating monolayer in which cells move laterally, and a stable monolayer in which cells are immobilized. Quantitative determination of cell motility was performed by analysing time-lapse cellular patterns. These computer-based geometrical analyses enabled us to divide monolayers into three groups: tensile stable monolayers, non-tensile stable monolayers and fluctuating monolayers, and this study therefore gives an insight into the way in which changing conformations of cells may be assayed.

Phosphatidylinositol 3,4,5-trisphosphate: an early mediator of insulin-stimulated sodium transport in A6 cells

2004 ◽  
Vol 287 (2) ◽  
pp. F319-F328 ◽  
Author(s):  
Nicolas Markadieu ◽  
Daniel Blero ◽  
Alain Boom ◽  
Christophe Erneux ◽  
Renaud Beauwens
Keyword(s):  
Sodium Transport ◽  
Cell Monolayer ◽  
Sodium Reabsorption ◽  
Na Channel ◽  
Insulin Stimulation ◽  
Cell Monolayers ◽  
A6 Cells ◽  
Cell Extracts ◽  
Pkb Phosphorylation ◽  
Stimulation Of

Insulin stimulates sodium transport across A6 epithelial cell monolayers. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was suggested as an early step in the insulin-stimulated sodium reabsorption (Ref. 35). To establish that the stimulation of the PI 3-kinase signaling cascade is causing stimulation of apical epithelial Na channel, we added permeant forms of phosphatidylinositol (PI) phosphate (P) derivatives complexed with a histone carrier to A6 epithelium. Only PIP3 and PI( 3 , 4 )P2 but not PI( 4 , 5 )P2 stimulated sodium transport, although each of them penetrated into A6 cell monolayers as assessed using fluorescent permeant phosphoinositides derivatives. By Western blot analysis of A6 cell extracts, the inositol 3-phosphatase PTEN and the protein kinase B PKB were both detected. To further establish that the stimulation of sodium transport induced by insulin is related to PIP3 levels, we transfected A6 cells with human PTEN cDNA and observed a 30% decrease in the natriferic effect of insulin. Similarly, the increase in sodium transport observed by addition of permeant PIP3 was also reduced by 30% in PTEN-overexpressing cells. PKB, a main downstream effector of PI 3-kinase, was phosphorylated at both Thr 308 and Ser 473 residues upon insulin stimulation of the A6 cell monolayer. PKB phosphorylation in response to insulin stimulation was reduced in PTEN-overexpressing cells. Permeant PIP3 also increased PKB phosphorylation. Taken together, the present results establish that the d-3-phosphorylated phosphoinositides PIP3 and PI( 3 , 4 )P2 mediate the effect of insulin on sodium transport across A6 cell monolayers.

OnBlastocystissecreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers

Parasitology ◽  
2016 ◽  
Vol 143 (13) ◽  
pp. 1713-1722 ◽  
Author(s):  
C. NOURRISSON ◽  
I. WAWRZYNIAK ◽  
A. CIAN ◽  
V. LIVRELLI ◽  
E. VISCOGLIOSI ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Proteolytic Enzymes ◽  
Cell Monolayer ◽  
Cysteine Proteases ◽  
Cell Permeability ◽  
Whole Genome Sequence ◽  
Intestinal Cell ◽  
Pathogenic Potential ◽  
Cell Monolayers ◽  
Culture Supernatants

SUMMARYBlastocystisspp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects.In silicoanalysis of the whole genome sequence ofBlastocystissubtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified inBlastocystissubtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to eitherBlastocystisculture supernatants or each recombinant protease. BothBlastocystisculture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis ofBlastocystisby increasing intestinal cell permeability.

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