Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes

Priesnitz, Christian; Sperber, Saskia; Garg, Richa; Orsini, Malina; Fatima, Nosheen

doi:10.1007/s10616-014-9821-1

Cited by 4 publications

(5 citation statements)

References 19 publications

(16 reference statements)

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“…The specific binding of Aβ42 was calculated by subtracting the fluorescence of cells in the absence of Aβ42 from that of fluorescence in the presence of varying concentrations of Aβ42. The data were analyzed using Image J software as described in Priesnitz et al [26].…”

Section: Aβ-rage Interaction In Transfected Cell Linementioning

confidence: 99%

G82S RAGE polymorphism influences amyloid-RAGE interactions relevant in Alzheimer’s disease pathology

2020

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Receptor for advanced glycation end products (RAGE) has been implicated in the pathophysiology of Alzheimers disease(AD) due to its ability to bind amyloid-beta (Aβ42) and mediate inflammatory response. G82S RAGE polymorphism is associated with AD but the molecular mechanism for this association is not understood. Our previous in silico study indicated a higher binding affinity for mutated G82S RAGE, which could be caused due to changes in N linked glycosylation at residue N81. To confirm this hypothesis, in the present study molecular dynamics (MD) simulations were used to simulate the wild type (WT) and G82S glycosylated structures of RAGE to identify the global structural changes and to find the binding efficiency with Aβ42 peptide. Binding pocket analysis of the MD trajectory showed that cavity/binding pocket in mutant G82S glycosylated RAGE variants is more exposed and accessible to external ligands compared to WT RAGE, which can enhance the affinity of RAGE for Aβ. To validate the above concept, an in vitro binding study was carried using SHSY5Y cell line expressing recombinant WT and mutated RAGE variant individually to which HiLyte Fluor labeled Aβ42 was incubated at different concentrations. Saturated binding kinetics method was adopted to determine the K d values for Aβ42 binding to RAGE. The K d value for Aβ42- WT and Aβ42-mutant RAGE binding were 92±40 nM (95% CI-52 to 152nM; R 2 -0.92) and 45±20 nM (95% CI -29 to 64nM; R 2 -0.93), respectively. The K d value of <100nM observed for both variants implicates RAGE as a high-affinity receptor for Aβ42 and mutant RAGE has higher affinity compared to WT. The alteration in binding affinity is responsible for activation of the inflammatory pathway as implicated by enhanced expression of TNFα and IL6 in mutant RAGE expressing cell line which gives a mechanistic view for the G82S RAGE association with AD.

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Section: Aβ-rage Interaction In Transfected Cell Linementioning

confidence: 99%

G82S RAGE polymorphism influences amyloid-RAGE interactions relevant in Alzheimer’s disease pathology

2020

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“…Other quality criteria included typical hepatocytes morphology with binucleated cells, cell contacts, and polarity which can be seen in cultures of both rat types irrespective of age groups. We used a previously described method to calculate the cell size . It was observed that the SD PRH were slightly larger in size (supplementary Table S6).…”

Section: Resultsmentioning

confidence: 99%

“…Cells were stained for 10 min with Calcein AM dissolved in WME in a final concentration of 4 μg/mL. The method for cell counting was established in our laboratory and is described in detail elsewhere …”

Section: Methodsmentioning

confidence: 99%

“…We used a previously described method to calculate the cell size. 16 It was observed that the SD PRH were slightly larger in size (supplementary Table S6). For both rat types there was no significant difference in the hepatocytes viability for both age groups from 12 to 24 h.…”

Section: Prh Viabilitymentioning

confidence: 98%

“…The method for cell counting was established in our laboratory and is described in detail elsewhere. 16…”

Section: Cell Viability Determinationmentioning

confidence: 99%

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Hepatocytes of Wistar and Sprague Dawley rats differ significantly in their central metabolism

Garg

Heinzle²,

Fatima³

2017

J of Cellular Biochemistry

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Wistar and Sprague-Dawley (SD) rats are most commonly used experimental rats. They have similar genetic background and are therefore, not discriminated in practical research. In this study, we compared metabolic profiles of Wistar and SD rat hepatocytes from middle (6 months) and old (23 months) age groups.Principle component analysis (PCA) on the specific uptake and production rates of amino acids, glucose, lactate and urea indicated clear differences between Wistar and SD rat hepatocytes. SD rat hepatocytes showed higher uptake rates of various essential and non-essential amino acids, particularly in early culture phases (0-12 h) compared to later phases (12-24 h). SD hepatocytes seem to be more sensitive to isolation procedure and in vitro culture requiring more amino acids for cellular maintenance and repair. Major differences between Wistar and SD rat hepatocytes were observed for glucose and branched chain amino acid metabolism. We conclude that the observed differences in the central carbon metabolism of isolated hepatocytes from these two rats should be considered when using one or the other rat type in studies on metabolic effects or diseases such as diabetes or obesity. K E Y W O R D Saging, amino acid metabolism, glucose metabolism, metabolic profiles, rat hepatocytes, RRID:Rats are frequently used laboratory animals for research owing to their easy maintenance, short gestation period and life span. Wistar and Sprague-Dawley (SD) are the two most common outbred rats in the laboratory and are often used in toxicological and aging studies. 1 Wistar rats are commonly used in Europe and SD rats are generally preferred in the US. 2 Wistar and SD rats have been used to study metabolic diseases like insulin resistance in type 2 diabetes and high fat diet induced obesity. [3][4][5][6] Differences between Wistar and SD rats regarding food intake, growth rate, hormone levels, 7 as well as tumor genesis 8 are known. Recently, a GC-MS based urinary metabolomics study revealed differences between the two rats under fasting and fed conditions and upon exposure to ethanol. 9 Another study, using nuclear magnetic resonance (NMR) based metabonomics showed differences in metabolite profiles in urine samples of these two types of rats. 10 Variations in the expression of CYP 450 1A and 3A enzymes have been described. 11 Differences between these two rats have also been observed in the manifestation of neurotoxicity 2 and drug toxicity. 12,13,14 Since these two rat strains are also commonly used in in vitro studies, a detailed study on the metabolic profiles of isolated hepatocytes in culture from these two rat strains would be very helpful.

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Cell density detection based on a microfluidic chip with two electrode pairs

2022

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Cell density detection is usually the counting of cells in certain volume of liquid, which is an important process in the eld of biomedicine. The Coulter counting method has been an important method for biological cell detection and counting for decades. In this paper, a micro uidic chip based on two electrode pairs is designed, which uses the Coulter principle to detect the ow rate of uncertain volume liquid and count the cells, and then calculate the cell density by statistics. When the cell passes through the sensor channel formed by the electrode pair on the chip, the impedance will change between the electrodes. This phenomenon has been proved by experiments. The designed chip has the advantages of simple structure, small size and low manufacturing cost. The cell density detection method proposed in this article is of great signi cance to the research in the eld of biological cell detection and development of related medical devices.

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Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes

Cited by 4 publications

References 19 publications

G82S RAGE polymorphism influences amyloid-RAGE interactions relevant in Alzheimer’s disease pathology

G82S RAGE polymorphism influences amyloid-RAGE interactions relevant in Alzheimer’s disease pathology

Hepatocytes of Wistar and Sprague Dawley rats differ significantly in their central metabolism

Cell density detection based on a microfluidic chip with two electrode pairs

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