scholarly journals The Petri Dish-N2B27 Culture Condition Maintains RPE Phenotype by Inhibiting Cell Proliferation and mTOR Activation

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Hui Lou ◽  
Chunpin Lian ◽  
Fanjun Shi ◽  
Liqun Chen ◽  
Sicheng Qian ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Proliferation ◽  
Growth Medium ◽  
Culture Condition ◽  
Petri Dish ◽  
High Density ◽  
High Yield ◽  
Low Density ◽  
Rpe Cells ◽  
Petri Dishes

Objective. To develop a method for the rapid isolation of rat RPE cells with high yield and maintain its epithelial state in modified culture system. Methods. The eyeballs were incubated with dispase. The retina was isolated with RPE attached and cut into several pieces. Following a brief incubation in growth medium, large RPE sheets can be harvested rapidly. RPE cells were divided into four groups and cultured for several weeks, that is, (1) in cell culture dishes with 10% FBS containing medium (CC dish-FBS), (2) in petri dishes with 10% FBS containing medium (Petri dish-FBS), (3) in cell culture dishes with N2 and B27 containing medium (CC dish-N2B27), and (4) in petri dishes with N2 and B27 containing medium (Petri dish-N2B27). Morphological and biological characteristics were investigated using light microscopy, Q-PCR, and western blot. Results. The retina would curl inwardly during the growth medium incubation period, releasing RPE sheets in the medium. Compared with low density group (5,000 cells/cm2), RPE cells plated at high density (15,000 cells/cm2) can maintain RPE morphology for a more extended period. Meanwhile, plating RPE cells at low density significantly reduced the expression of RPE cell type-specific genes (RPE65, CRALBP, and bestrophin) and increased the expression of EMT-related genes (N-cadherin, fibronectin, and α-SMA), in comparison with the samples from the high density group. The petri dish culture condition reduced cell adhesion and thus inhibited RPE cell proliferation. As compared with other culture conditions, RPE cells in the petri dish-N2B27 condition could maintain RPE phenotype with increased expression of RPE-specific genes and decreased expression of EMT-related genes. The AKT/mTOR pathway was also decreased in petri dish-N2B27 condition. Conclusion. The current study provided an alternative method for easy isolation of RPE cells with high yield and maintenance of its epithelial morphology in the petri dish-N2B27 condition.

scholarly journals The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient

Cell Medicine ◽  
2017 ◽  
Vol 9 (1-2) ◽  
pp. 45-51 ◽  
Author(s):  
Chika Miyagi-Shiohira ◽  
Naoya Kobayashi ◽  
Issei Saitoh ◽  
Masami Watanabe ◽  
Yasufumi Noguchi ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Density Gradient ◽  
Stimulation Index ◽  
High Density ◽  
Steel Pipe ◽  
High Yield ◽  
Low Density ◽  
Pancreatic Islet Transplantation ◽  
Continuous Density ◽  
Stainless Steel Pipe

Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. The most common method of islet purification is density gradient centrifugation using a COBE 2991 cell processor. However, this method can damage islets mechanically through its high shearing force. We recently reported that a new purification method using large plastic bottles effectively achieves a high yield of islets from the porcine pancreas. In the present study, we evaluated the methods of making a continuous density gradient. The gradient was produced with a gradient maker and two types of candy cane-shaped stainless steel pipes. One method was to use a “bent-tipped” stainless steel pipe and to load from a high-density solution to a low-density solution, uploading the stainless steel pipe. The other method was to use a regular stainless steel pipe and to load from a low-density solution to a high-density solution, leaving the stainless steel pipe in place. There were no significant differences between the two solutions in terms of the islet yield, rate of viability or purity, score, or the stimulation index after purification. Furthermore, there were no differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

scholarly journals The relation of platelet density to platelet age: survival of low- and high-density 111indium-labeled platelets in baboons

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 386-393
Author(s):  
B Savage ◽  
PR McFadden ◽  
SR Hanson ◽  
LA Harker
Keyword(s):  
Specific Radioactivity ◽  
High Density ◽  
High Yield ◽  
Low Density ◽  
Density Gradients ◽  
Continuous Linear ◽  
Survival Times ◽  
Density Fractions ◽  
Age Related

The relationship between platelet density and platelet age has been studied using continuous linear Percoll density gradients and 111In- labeling of autologous platelets in baboons. To investigate changes in platelet density during senescence in the circulation, baboons were infused with 111In-labeled autologous platelets, and blood was collected at one hour postinfusion and twice daily thereafter for six days. Platelets were isolated from these samples in high yield (greater than 95%) and separated in continuous linear Percoll density gradients following density equilibrium centrifugation. Although at one hour postinfusion the density distribution of radiolabeled platelets coincided closely with the distribution of the total platelet population, a detectable symmetrical shift toward higher densities was observed after five days. The relative specific radioactivity (RSR) of high-density platelets (1.064 to 1.067 g/mL) decreased at a slower rate than that of the total platelet population (platelets of all densities), whereas the RSR of low-density platelets (1.053 to 1.056 g/mL) showed a more immediate and rapid decrease. These results give rise to one of two interpretations: (1) low-density platelets have a shorter survival time than more dense platelets and are therefore cleared from the circulation at a faster rate, or (2) platelets of all densities increase in density upon aging in the circulation. To determine the explanation for changing RSR of different density fractions we studied the in vivo disappearance characteristics of low- and high-density 111In-labeled platelets. There were no significant differences between the mean survival times of low-density platelets (5.0 +/- 0.49 days, +/- 1 SD, n = 6), high-density platelets (4.9 +/- 0.56 days, n = 6), or control platelets representing platelets of all densities (4.9 +/- 0.38 days, n = 6). Although a slight increase in the density of all platelets during platelet senescence is indicated by these studies, we conclude that platelet density heterogeneity is not primarily a consequence of age-related changes in platelet density.

Evidence for ERK1/2 phosphorylation controlling contact inhibition of proliferation in Madin-Darby canine kidney epithelial cells

2004 ◽  
Vol 287 (2) ◽  
pp. C432-C439 ◽  
Author(s):  
Shixiong Li ◽  
Edward R. Gerrard ◽  
Daniel F. Balkovetz
Keyword(s):  
Cell Proliferation ◽  
Cyclin D1 ◽  
Mdck Cell ◽  
Mdck Cells ◽  
High Density ◽  
Contact Inhibition ◽  
Low Density ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Increasing cell density arrests epithelial cell proliferation by a process termed contact inhibition. We investigated mechanisms of contact inhibition using a model of contact-inhibited epithelial cells. Hepatocyte growth factor (HGF) treatment of contact-inhibited Madin-Darby canine kidney (MDCK) cells stimulated cell proliferation and increased levels of phosphorylated ERK1/2 (phospho-ERK1/2) and cyclin D1. MEK inhibitors PD-98059 and U0126 inhibited these HGF-dependent changes, indicating the dependence on phosphorylation of ERK1/2 during HGF-induced loss of contact inhibition. In relation to contact-inhibited high-density cells, low-density MDCK cells proliferated and had higher levels of phospho-ERK1/2 and cyclin D1. PD-98059 and U0126 inhibited low-density MDCK cell proliferation. Trypsinization of high-density MDCK cells immediately increased phospho-ERK1/2 and was followed by a transient increase in cyclin D1 levels. Reformation of cell junctions after trypsinization led to decreases in phospho-ERK1/2 and cyclin D1 levels. High-density MDCK cells express low levels of both cyclin D1 and phospho-ERK1/2, and treatment of these cells with fresh medium containing HGF but not fresh medium alone for 6 h increased phospho-ERK1/2 and cyclin D1 levels compared with cells without medium change. These data provide evidence that HGF abrogates MDCK cell contact inhibition by increasing ERK1/2 phosphorylation and levels of cyclin D1. These results suggest that in MDCK cells, contact inhibition of cell proliferation in the presence of serum occurs by cell density-dependent regulation of ERK1/2 phosphorylation.

scholarly journals The relation of platelet density to platelet age: survival of low- and high-density 111indium-labeled platelets in baboons

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 386-393 ◽  
Author(s):  
B Savage ◽  
PR McFadden ◽  
SR Hanson ◽  
LA Harker
Keyword(s):  
Specific Radioactivity ◽  
High Density ◽  
High Yield ◽  
Low Density ◽  
Density Gradients ◽  
Continuous Linear ◽  
Survival Times ◽  
Density Fractions ◽  
Age Related

Abstract The relationship between platelet density and platelet age has been studied using continuous linear Percoll density gradients and 111In- labeling of autologous platelets in baboons. To investigate changes in platelet density during senescence in the circulation, baboons were infused with 111In-labeled autologous platelets, and blood was collected at one hour postinfusion and twice daily thereafter for six days. Platelets were isolated from these samples in high yield (greater than 95%) and separated in continuous linear Percoll density gradients following density equilibrium centrifugation. Although at one hour postinfusion the density distribution of radiolabeled platelets coincided closely with the distribution of the total platelet population, a detectable symmetrical shift toward higher densities was observed after five days. The relative specific radioactivity (RSR) of high-density platelets (1.064 to 1.067 g/mL) decreased at a slower rate than that of the total platelet population (platelets of all densities), whereas the RSR of low-density platelets (1.053 to 1.056 g/mL) showed a more immediate and rapid decrease. These results give rise to one of two interpretations: (1) low-density platelets have a shorter survival time than more dense platelets and are therefore cleared from the circulation at a faster rate, or (2) platelets of all densities increase in density upon aging in the circulation. To determine the explanation for changing RSR of different density fractions we studied the in vivo disappearance characteristics of low- and high-density 111In-labeled platelets. There were no significant differences between the mean survival times of low-density platelets (5.0 +/- 0.49 days, +/- 1 SD, n = 6), high-density platelets (4.9 +/- 0.56 days, n = 6), or control platelets representing platelets of all densities (4.9 +/- 0.38 days, n = 6). Although a slight increase in the density of all platelets during platelet senescence is indicated by these studies, we conclude that platelet density heterogeneity is not primarily a consequence of age-related changes in platelet density.

scholarly journals Yield of Saffron (Crocus sativus) Under Different Corm Densities

2019 ◽  
Vol 11 (8) ◽  
pp. 183
Author(s):  
Abdel Kader El Hajj ◽  
Sally Moustafa ◽  
Soha Oleik ◽  
Vera Telj ◽  
Nour Taha ◽  
...  
Keyword(s):  
Cultural Practices ◽  
Agricultural Research ◽  
Block Design ◽  
High Density ◽  
Crocus Sativus ◽  
High Yield ◽  
Low Density ◽  
Yield Attributes ◽  
Alternative Crop ◽  
The Impact

Due to its valuable properties, Saffron is considered one of the most expensive spices. Saffron was introduced to Lebanon in 2000 and promoted as alternative crop to Canabis Sativa. There has been little research for saffron on a variety of subjects, in particular on cultural practices. The aim of this study is to investigate the impact of corm density on Saffron yield attributes. The study included three corm densities that were organized in randomized complete block design with three replicates; Low(LD)-25 corms m-2, 20 × 20 cm spacing; Moderate (MD)-44 corms m-2, 15 × 15 cm spacing; High (HD)-100 corms m-2, 10 × 10 cm spacing. The study was conducted at the Lebanese agricultural research institute (LARI) in Lebaa station for four consecutive years. Results showed a steadily significant high yield of flowers m-2 at high density compared to low density for four years .In contrast, the number of flowers (NF)/100 corm clusters (CC) was significantly increased at low density in last two years compared to high density. Treatments showed no significant effect of the main compounds of air-dried stigmas (Crocin, Picrocrocin and Safranal). Fresh (FWSS) and dry (DWSS) weight of single stigma was affected by year showing a significant change from year to year. These results revealed that success of long-term cultivation can be achieved by planting saffron corm at moderate and low densities.

Quantitative defect studies in semiconductor TEM samples prepared by low angle ion milling

1995 ◽  
Vol 53 ◽  
pp. 512-513
Author(s):  
L. Mulestagno ◽  
J.C. Holzer ◽  
P. Fraundorf
Keyword(s):  
Sample Preparation ◽  
Milling Time ◽  
High Density ◽  
Low Density ◽  
Ion Milling ◽  
High Quality ◽  
Variable Angle ◽  
Major Disadvantage ◽  
Induced Defects

Due to the wealth of information, both analytical and structural that can be obtained from it TEM always has been a favorite tool for the analysis of process-induced defects in semiconductor wafers. The only major disadvantage has always been, that the volume under study in the TEM is relatively small, making it difficult to locate low density defects, and sample preparation is a somewhat lengthy procedure. This problem has been somewhat alleviated by the availability of efficient low angle milling.Using a PIPS® variable angle ion -mill, manufactured by Gatan, we have been consistently obtaining planar specimens with a high quality thin area in excess of 5 × 104 μm2 in about half an hour (milling time), which has made it possible to locate defects at lower densities, or, for defects of relatively high density, obtain information which is statistically more significant (table 1).

Scanning Electron Microscopic and Electrophoretic Observations on Barium Sulphate Used to Adsorb Clotting Factors

1975 ◽  
Vol 33 (02) ◽  
pp. 256-270
Author(s):  
R. M Howell ◽  
S. L. M Deacon
Keyword(s):  
Electron Microscopy ◽  
Factor Xa ◽  
Barium Sulphate ◽  
High Density ◽  
Low Density ◽  
Factor X ◽  
Clotting Factors ◽  
Electron Microscopic ◽  
Complementary Techniques ◽  
Scanning Electron

SummaryElectron microscopy and particle electrophoresis were found to be complementary techniques with which to complete the physical data from an earlier study on barium sulphates used to adsorb clotting factors from serum. The differences revealed by scanning electron microscopy (S. E. M.) in the physical shape of low and high density grades of barium sulphate particles appear to be of greater significance than charge as expressed by electrophoretic mobility, in determining whether or not precursor or preformed factor Xa is eluted.This conclusion was based on the finding that at pH values close to 7, where the adsorption from serum occurs, all samples with the exception of natural barytes were uncharged. However as the high-density, or soil-grade, was found by S. E. M. to consist of large solid crystals it was suggested that this shape might induce activation of factor X as a result of partial denaturation and consequent unfolding of the adsorbed protein. In contrast, uptake of protein into the centre of the porous aggregates revealed by S. E. M. pictures of low-density or X-ray grade barium sulphate may afford protection against denaturation and exposure of the enzyme site.The porous nature of particles of low-density barium sulphate compared with the solid crystalline forms of other grades accounts not only for its lower bulk density but also for its greater surface/gram ratio which is reflected by an ability to adsorb more protein from serum.Neither technique produced evidence from any of the samples to indicate the presence of stabilising agents sometimes used to coat particles in barium meals.

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