The Effects of Trypan Blue on Chick Embryos Cultured in vitro

Development ◽  
1960 ◽  
Vol 8 (1) ◽  
pp. 1-5
Author(s):  
Leela Mulherkar
Keyword(s):  
Trypan Blue ◽  
Teratogenic Effect ◽  
Chick Embryos ◽  
Cent Solution ◽  
Stage Of Development ◽  
Vital Dye ◽  
Amphibian Embryos ◽  
Set Up ◽  
Rats And Mice

Various malformations in rats and mice have been observed to be caused by administration of the vital dye, Trypan blue (Gillman, Gilbert, Gillman, & Spence, 1948; Fox & Goss, 1956, 1957; Hamburgh, 1952, 1954; Waddington & Carter, 1952, 1953). Recently Waddington & Perry (1956) reported a teratogenic effect of Trypan blue on Amphibian embryos. The present paper deals with the effects of this dye on cultured chick embryos. Hen's eggs were incubated at 38°C. to the desired stage of development. Cultures were set up according to the ring technique described by New (1955). Precautions to maintain sterile conditions were observed throughout. An aqueous 1 per cent, solution of Gurr's vital Trypan blue was diluted with Pannett Compton saline to 0·05 per cent., which had been found to be a suitable concentration. A few drops of the dye solution were placed on the upper surface of the treated blastoderms.

Teratogenic Effects of Trypan Blue on Amphibian Embryos

Development ◽  
1956 ◽  
Vol 4 (2) ◽  
pp. 110-118
Author(s):  
C. H. Waddington ◽  
M. M. Perry
Keyword(s):  
Trypan Blue ◽  
Teratogenic Effects ◽  
Pregnant Rat ◽  
Vital Dye ◽  
Amphibian Embryos ◽  
Mammalian Embryos

Considerable interest has been aroused by the report of Gillman, Gilbert, Gillman, & Spence (1948) that the vital dye trypan blue when injected into the pregnant rat brings about the appearance of various abnormalities in the offspring. The mammal embryo is notoriously difficult to attack experimentally, and trypan blue therefore seems to provide a valuable tool which, moreover, might throw light on the mechanisms of the important effects which are known in some cases to be produced on the foetus by pathological states in the mother. Further studies on the effects of injected trypan blue on mammalian embryos have therefore been made by Hamburgh (1952, 1954), Waddington & Carter (1952, 1953), and Murakami and his collaborators (1952, 1954), all of whom worked on the mouse, and by Harm (1954) who studied the rabbit. All these authors agree, on the whole, in the type of results which they have obtained, although there are some minor differences between their reports which will be considered later.

scholarly journals Teratogenic Effect of Trypan Blue on Rat Embryos cultivated in vitro

Nature ◽  
1965 ◽  
Vol 206 (4984) ◽  
pp. 637-637 ◽  
Author(s):  
MYRON M. TURBOW
Keyword(s):  
Trypan Blue ◽  
Teratogenic Effect ◽  
Rat Embryos

A Heparin-coated Circuit Maintains Platelet Aggregability in Response to Shear Stress in an In Vitro Model of Cardiopulmonary Bypass

1998 ◽  
Vol 80 (09) ◽  
pp. 437-442 ◽  
Author(s):  
I. Hioki ◽  
K. Onoda ◽  
T. Shimono ◽  
H. Shimpo ◽  
K. Tanaka ◽  
...  
Keyword(s):  
Shear Stress ◽  
Cardiopulmonary Bypass ◽  
Membrane Skeleton ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Platelet Aggregability ◽  
Vitro Model ◽  
Platelet Defects ◽  
Set Up

SummaryAlterations in platelet aggregability may play a role in the pathogenesis of qualitative platelet defects associated with cardiopulmonary bypass (CPB). We circulated fresh heparinized whole blood through tubing sets coated with heparin (C group, n = 10) and through non-coated sets (N group, n = 10) as a simulated CPB circuit. Shear stress (108 dyne/cm2)-induced platelet aggregation (hSIPA), plasma von Willebrand factor (vWF) activity and platelet glycoprotein (GP) Ib expression were measured, before, during, and after this in vitro set up of circulation. In the two groups, the extent of hSIPA significantly decreased during circulation and was partially restored after circulation. Decreases in the extent of hSIPA were significantly less with use of heparin-coated circuits. There was an equivalent reduction in plasma vWF activity, in the two groups. Expression of platelet surface GP Ib decreased significantly during circulation and recovered after circulation. Reduction of surface GP Ib expression during circulation was significantly less in the C group than that in the N group. Decrease in surface GP Ib expression correlated (r = 0.88 in either group) with the magnitude of hSIPA, in the two groups. The progressive removal of surface GP Ib was mainly attributed to redistribution of GP Ib from the membrane skeleton into the cytoskeleton. Our observations suggest that use of heparin-coated circuits partly blocks the reduction of hSIPA, as a result of a lesser degree of redistribution of GP Ib.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Plant Biotechnology Workshop for High School Students

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 504e-504
Author(s):  
Erika Szendrak ◽  
Paul E. Read ◽  
Jon S. Miller
Keyword(s):  
High School ◽  
High School Students ◽  
Medical Science ◽  
Plant Biotechnology ◽  
Plant Science ◽  
High School Biology ◽  
School Students ◽  
Subsequent Transformation ◽  
Set Up

Modern aspects of many subjects (e.g., computer science and some aspects of medical science) are now taught in many high schools, but the plant sciences are often given short shrift. A collaboration was therefore established with a high school biology program in which pilot workshops could be developed to enable advanced students to gain insights into modern plant science techniques. A successful example is the workshop on plant biotechnology presented in this report. This workshop is simple and flexible, taking into account that most high school biology laboratories and classrooms are not set up for sophisticated plant science/biotechnology projects. It is suitable for from 10 to 30 students, depending upon space and facilities available. Students work in pairs or trios, and learn simple disinfestation and transfer techniques for micropropagation and potential subsequent transformation treatments. Students gain insights into: sterile technique and hygiene; plant hormones and their physiological effects; plant cell, tissue and organ culture; the influence of environmental factors on response of cells and tissues cultured in vitro; and an understanding of the phenomenon of organogenesis and resulting plant growth and development. This workshop has been tested on several classes of students and following analysis, several refinements were included in subsequent iterations. Results of the students' experiments have been positive and instructive, with student learning outcomes above expectations. Further details of the workshop techniques and approach will be presented.

scholarly journals Multiple screening approaches reveal HDAC6 as a novel regulator of glycolytic metabolism in triple-negative breast cancer

2021 ◽  
Vol 7 (3) ◽  
pp. eabc4897
Author(s):  
Catríona M. Dowling ◽  
Kate E. R. Hollinshead ◽  
Alessandra Di Grande ◽  
Justin Pritchard ◽  
Hua Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Histone Deacetylase Inhibitors ◽  
Triple Negative ◽  
Mitochondrial Apoptosis ◽  
Glycolytic Metabolism ◽  
Set Up ◽  
Screening Approaches

Triple-negative breast cancer (TNBC) is a subtype of breast cancer without a targeted form of therapy. Unfortunately, up to 70% of patients with TNBC develop resistance to treatment. A known contributor to chemoresistance is dysfunctional mitochondrial apoptosis signaling. We set up a phenotypic small-molecule screen to reveal vulnerabilities in TNBC cells that were independent of mitochondrial apoptosis. Using a functional genetic approach, we identified that a “hit” compound, BAS-2, had a potentially similar mechanism of action to histone deacetylase inhibitors (HDAC). An in vitro HDAC inhibitor assay confirmed that the compound selectively inhibited HDAC6. Using state-of-the-art acetylome mass spectrometry, we identified glycolytic substrates of HDAC6 in TNBC cells. We confirmed that inhibition or knockout of HDAC6 reduced glycolytic metabolism both in vitro and in vivo. Through a series of unbiased screening approaches, we have identified a previously unidentified role for HDAC6 in regulating glycolytic metabolism.

scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

scholarly journals EPEN-33. PHARMACOGENOMICS REVEALS SYNERGISTIC INHIBITION OF ERBB2 AND PI3K SIGNALING AS A THERAPEUTIC STRATEGY FOR EPENDYMOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii314-iii314
Author(s):  
David Pauck ◽  
Eunice Paisana ◽  
Rita Cascão ◽  
Sevgi Sarikaya-Seiwert ◽  
Viktoria Marquardt ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Single Agent ◽  
Pediatric Brain Tumor ◽  
Pediatric Brain Tumors ◽  
Primary Diagnosis ◽  
High Throughput Drug Screening ◽  
Established Cell ◽  
Set Up ◽  
Pediatric Brain

Abstract Subgroups of ependymoma, especially RELA fusion-positive and posterior fossa type A tumors, are associated with poor prognosis. Curative therapeutic strategies have not yet been identified. We set up a high-throughput drug screening (HTS) pipeline to evaluate clinically established compounds (n=196) in primary ependymoma cultures (n=12). As culturing ependymoma is challenging, assay miniaturization to 1536-well microplates emerged as a key feature to process HTS despite smallest cell numbers. DNA methylation profiling showed that entity and subgroup affiliation from primary diagnosis was maintained in primary cultures, as assessed through molecular neuropathology 2.0 based classification (MNP 2.0, Capper, D. et al., Nature, 2018). A comparison of HTS data of ependymoma and other pediatric brain tumor models (n=48) revealed a remarkable chemoresistance in vitro. However, we identified Neratinib, an irreversible ERBB2 inhibitor, as the most prominent candidate which was preferentially active in a subset of the investigated ependymoma cultures (n=5). Combinatory treatment with Copanlisib, a PI3K inhibitor, was able to overcome resistance to single agent treatment using Neratinib in established cell lines of ependymoma (n=3) and 2/4 primary cultures for which combinatory treatment could be tested. Finally, we validated efficacy of Neratinib combined with Copanlisib in mice bearing ependymoma xenografts which revealed significantly reduced tumor size compared to vehicle-treated animals. In summary, our study demonstrates that HTS may reveal targeted therapies for pediatric brain tumors. Specifically, we found a synergistic interaction of Neratinib and Copanlisib for treatment of ependymoma, thereby providing a novel therapeutic approach in an otherwise largely chemoresistant entity.

scholarly journals Using a roller pump for establishing extra-corporal membrane oxygenation (ECMO) – technical considerations for times of crisis

Perfusion ◽  
2021 ◽  
pp. 026765912199618
Author(s):  
Mirko Kaluza ◽  
Benjamin May ◽  
Torsten Doenst
Keyword(s):  
Flow Characteristics ◽  
Centrifugal Pumps ◽  
Roller Pump ◽  
Silicone Tubing ◽  
Heart Lung Machine ◽  
Surface Irregularities ◽  
Set Up ◽  
Flow Detection ◽  
Silicon Tubing

Objective: The COVID-19 pandemic requires thinking about alternatives to establish ECMO when often-limited hardware resources are exhausted. Heart-lung-machines may potentially be used for ECMO but contain roller pumps as compared to centrifugal pumps in ECMO-circuits. We here tested roller pumps as rescue pump for ECMO-establishment. Methods: We set up in vitro circuits on roller pumps from C5 heart-lung-machine with 5 l/minutes flow. In two series, we placed either PVC or silicon tubing for an ECMO circuit into the roller pump. We assessed the mechanical stress on the tubing (aiming to run the pump for at least 1 week), measured the temperature increase generated by the friction and assessed flow characteristics and its measurement in simulated situations resembling tube kinking and suction. Results: The roller pumps led to expected and unexpected adverse events. PVC tubing burst between 36 and 78 hours, while silicon tubing lasted for at least 7 days. At 7 days, the silicone tubing showed significant signs of roller pump wear visible on the outside. The inside, however, was free of surface irregularities. Using these tubings in a roller pump led to a remarkable increase in circuit temperature (PVC: +12.0°C, silicone +2.9°C). Kinking or suction on the device caused the expected dramatic flow reduction (as assessed by direct measurement) while the roller pump display continued to show the preset flow. The roller pump is therefore not able to reliably determine the true flow rate. Conclusion: Roller pumps with silicone tubing but not PVC tubing may be used for running ECMO circuits. Silicone tubing may endure the roller pump shear forces for up to 1 week. Thus, repeated tubing repositioning may be a solution. Circuit heating and substantial limitations in flow detection should increase attention if clinical use in situations of crisis is considered.

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