Best Practices for Authentication of Cell Lines to Ensure Data Reproducibility and Integrity

Cell line misidentification and contamination are major contributors to the reproducibility crisis in academic research. Authentication of cell lines provides assurances of the data generated; however, commercially available cells are often not subjected to rigorous identification testing. In this study, commercially available cell lines underwent testing to confirm cell identity and purity. The methods reported here outline the best practices for cell line authentication. Briefly, a commercially available primary rabbit aortic endothelial cell line was purchased for the intent of producing target proteins necessary for generating species-specific recombinant antibodies. These rabbit-specific antibodies would then be utilized for the development of in-house enzyme-linked immunosorbent assays (ELISA) to evaluate blood-based biomarkers of vascular injury after total-body irradiation. To authenticate the cell line, cell identity and purity were determined by single tandem repeat (STR) testing, flow cytometry, polymerase chain reaction (PCR), and cytochrome c oxidase subunit 1 (CO1) DNA Barcoding in-house and/or through commercial vendors. Fresh cells obtained from a New Zealand White rabbit (Charles River, Wilmington, DE) were used as a positive control. The results of STR and flow cytometry analyses indicated the cells were not contaminated with human or mouse cells, and that the cells were not of endothelial origin. PCR demonstrated that cells were also not of rabbit origin, which was further confirmed by a third-party vendor. An unopened vial of cells was submitted to another vendor for CO1 DNA Barcoding analysis, which identified the cells as being purely of bovine origin. Results revealed that despite purchase through a commercial vendor, the cell line marketed as primary rabbit aortic endothelial cells were of bovine origin. Purity analysis found cells were misidentified rather than contaminated. Further investigation to determine the cell type was not performed. The most cost-effective and efficient methodology for confirming cell line identity was found to be CO1 DNA Barcoding performed by a commercial vendor.

DISTRIBUTION OF GABA TRANSPORTER (GAT1) LEVELS IN THE BÖTZINGER COMPLEX AT THE EARLY STAGES OF POSTNATAL DEVELOPMENT IN RATS WITH PRENATAL SEROTONIN DEFICIENCY

Цель работы - исследование распределения уровня GAT-транспортера ГАМК в комплексе Бетцингера на разных сроках раннего постнатального развития крыс в норме и при пренатальном дефиците серотонина. Материал и методы. Работа проведена на лабораторных крысах линии Wistar. Снижение уровня эндогенного серотонина в эмбриональный период осуществляли методом ингибирования триптофан-гидроксилазы пара-хлорфенилаланином (пХФА). Выявление транспортного белка GAТпроводили посредством иммуногистохимической реакции с использованием первичных кроличьих поликлональных антител anti-GABA transporter1 (AbCam, Великобритания). Мозг исследовали на 5-, 10-е и 20-е сутки постнатального развития. Результаты. В комплексе Бетцингера на ранних сроках постнатального развития у контрольных животных отмечено колебание уровня GAT-транспортера ГАМК. На 1-й неделе жизни уровень GATбыл высоким как в сети отростков и терминалей, так и в синапсах. В течение 2-й недели жизни уровень GATснижался, а к концу 3-й недели - повышался вновь, достигая исходного уровня. Дефицит серотонина в пренатальный период вызывал у подопытных животных существенное увеличение уровня GATв нейропиле комплекса Бетцингера на всех изученных сроках постнатального развития. Выводы. Пренатальный дефицит серотонина приводит к существенному повышению уровня GAT-транспортера ГАМК в ранние сроки постнатального развития, что может приводить к изменению трансмиссии ГАМК и, как следствие, к нарушению баланса тормозных и возбуждающих эффектов в дыхательном ядре. Objective - to study the distribution of GABA transporter 1 (GAT) levels in the Bötzinger complex at the early stages of postnatal development in rats with prenatal serotonin deficiency. Materials and methods. The work was carried out on Wistar line laboratory rats. To reduce the level of endogenous serotonin in the embryonic period, the method of tryptophan hydroxylase inhibition by para-chlorophenylalanine (PCPA) (Sigma, USA) was used. The GAT1 transport protein was detected by immunohistochemical reaction with anti-GABA transporter1 primary rabbit polyclonal antibodies (AbCam, UK). The brain was examined on the 5, 10 and 20 day of postnatal development. Results. At the early stages of postnatal development, a fluctuation in the GAT1 level of the GABA transporter was noted in the Bötzinger complex of control animals. In the first postnatal week, the GAT level was high both in the network of neuronal processes and terminals, and in synapses. During the 2 week of life, the GAT1 level decreased, and by the end of the 3 week it increased again, reaching the initial level. Deficiency of serotonin in the prenatal period caused a significant increase in the level of GAT in the neuropil of the Bötzinger complex in experimental animals at all studied stages of postnatal development. Conclusions. Prenatal deficiency of serotonin leads to a significant increase in the GAT1 level at the early stages of postnatal development, which can lead to a change in the GABA transmission, and, as a result, to a disturbance in the balance of inhibitory and stimulatory effects in the respiratory nuclei.

PREPARATION AND PROPERTIES OF MURINE ANTI-IDIOTYPIC MONOCLONAL ANTIBODIES RECOGNIZING PRIMARY RABBIT POLYCLONAL ANTIBODIES AGAINST MORPHINE DERIVATIVES

Anti-idiotypic antibodies (Ab2), according to the network theory of Jerne, are second-generation immunoglobulins that are produced against the idiotype of an antibody to a specific antigen. Despite the large number of works devoted to the study of the properties of these proteins, their role in the regulation of the immune system is not fully known. It may consist in maintaining or blocking a minimal immune response to the antigen. The study of Ab2 is of great practical and scientific importance. The special properties of Ab2, namely, the ability to partially reproduce the structure of the primary antigen and, upon immunization, induce the appearance of tertiary antibodies, which, like first-generation antibodies, can bind to the antigen, have found application in the development of Ab2-based vaccines, in particular, for the treatment of tumors. In view of the presence of a number of limitations on research related to psychoactive substances, the development of Ab2- based vaccines against drug addiction also seems promising. To example, anti-idiotypic antibodies obtained for this purpose possessing a cocaine-like structure are described in the literature. In this work, murine monoclonal anti-idiotypic antibodies (mAb2) mimicking the structure of various morphine derivatives were obtained. Rabbit polyclonal antibodies to the 6-hemisuccinyl derivative of morphine conjugated with bovine serum albumin isolated by affinity chromatography were used as primary antibodies for immunization. Four hybridoma clones were obtained as a result of the fusion of immunized mice lymphocytes with mouse Sp2/0 mouse myeloma cells by the Milstein-Köhler method. After growth in animals, mAb2 produced by hybridoma cells were affinity purified. We investigated the physicochemical and antigenic properties of the isolated antibodies. It was shown that the obtained mAb2 differ in immunological specificity, competing in different degree with morphine derivatives for binding to first-generation antibodies. We tested the possibility of using the obtained mAb2 as antigen analogues in the solid-phase enzyme-linked immunosorbent assay to determine the titer of primary antibodies against morphine in the blood serum of laboratory animals immunized with morphine derivatives. Based on the obtained anti-idiotypic antibodies, it is proposed to develop test systems to determine the serum opiate-specific antibodies in people after specific vaccination for therapeutic or prophylactic purposes to avoid the use of drugs as antigens immobilized on the solid phase in the analysis.

Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen

Melanocytes (MCs) are specialized cells that synthesize melanin within the melanosome. Cultured MCs are useful in order to study their role in relation to pigmentation. However, MC isolation is laborious and the obtained cells have a limited culture time. In this study, we transformed lentivirus-mediated simian virus 40 Large T (SV40-LT) into primary rabbit melanocytes (Pri RMCs) to establish an immortalized cell line. Morphologically, the immortalized RMCs (Im RMC) were indistinguishable from the Pri RMCs, and dendrites were visible following Dopa staining. No significant differences in cell proliferation or growth between immortalized and primary RMCs were observed. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 were detected by PCR, immunofluorescence staining, and western blot analysis. Through karyotype, soft agar, and tumorigenesis assays, the immortalized RMCs did not undergo malignant transformation. Our results show that Im RMCs can be used as a tool cell for future MC studies on the pigmentation mechanisms of fur animals.

Simvastatin prevents articular chondrocyte dedifferentiation induced by nitric oxide by inhibiting the expression of matrix metalloproteinases 1 and 13

In this study, we investigated whether simvastatin (SVT), a statin commonly prescribed to decrease cholesterol levels, might have a therapeutic effect in OA. Primary rabbit chondrocytes were pre-treated with SVT (50 μM), then treated with sodium nitroprusside (SNP; 1 mM), a donor of nitric oxide (NO) known as a pro-inflammatory mediator, and analyzed for the expression levels of type II collagen, SOX-9, aggrecan, matrix metalloproteinases (MMPs) 1, and 13. SNP increased NO generation in a dose-dependent manner, causing a loss of type II collagen and aggrecan indicative of chondrocyte dedifferentiation, which was inhibited by SVT. SVT also reversed the increase in MMP-1 and -13 and inhibited NO production and NO synthase expression induced by SNP in articular chondrocytes. Given that MMP-1 and -13 knockdown by siRNA increased the level of type II collagen in SNP-treated cells, our results show that SVT prevented NO-induced chondrocyte damage and dedifferentiation through downregulation of MMP expression. This study showed that SVT could attenuate the degradation of articular cartilage components, which is characteristic for OA, through inhibition of MMPs in NO-treated chondrocytes, suggesting that SVT may be a novel candidate therapeutic agent for the prevention and/or treatment of OA. Impact statement Dedifferentiation of chondrocytes is the main character of cartilage degradation. Therefore the understanding of chondrocytes dedifferentiation is essential for arthritis therapy. However, the molecular mechanism of cartilage destroy is mostly unknown. In this work we show that simvastatin (SVT) inhibits dedifferentiation by nitric oxide by blocking the expression of matrix metalloproteinases 1 and 13. These effects of SVT on dedifferentiation suggest that SVT may be used as a drug for the cure of arthritis.

Differential Impacts of Soybean and Fish Oils on Hepatocyte Lipid Droplet Accumulation and Endoplasmic Reticulum Stress in Primary Rabbit Hepatocytes

Parenteral nutrition-associated liver disease (PNALD) is a severe ailment associated with long-term parenteral nutrition. Soybean oil-based lipid emulsions (SOLE) are thought to promote PNALD development, whereas fish oil-based lipid emulsions (FOLE) are thought to protect against PNALD. This study aimed to investigate the effects of SOLE and FOLE on primary rabbit hepatocytes. The results reveal that SOLE caused significant endoplasmic reticulum (ER) and mitochondrial damage, ultimately resulting in lipid droplets accumulation and ER stress. While these deleterious events induce hepatocyte injury, FOLE at high doses cause only minor ER and mitochondrial damage, which has no effect on hepatic function. SOLE also significantly upregulated glucose-regulated protein 94 mRNA and protein expression. These data indicate that SOLE, but not FOLE, damage the ER and mitochondria, resulting in lipid droplets accumulation and ER stress and, finally, hepatocyte injury. This likely contributes to the differential impacts of SOLE and FOLE on PNALD development and progression.

Stimulation of gastric acid secretion by rabbit parietal cell A2B adenosine receptor activation

Adenosine modulates different functional activities in many cells of the gastrointestinal tract; some of them are believed to be mediated by interaction with its four G protein-coupled receptors. The renewed interest in the adenosine A2B receptor (A2BR) subtype can be traced by studies in which the introduction of new genetic and chemical tools has widened the pharmacological and structural knowledge of this receptor as well as its potential therapeutic use in cancer and inflammation- or hypoxia-related pathologies. In the acid-secreting parietal cells of the gastric mucosa, the use of various radioligands for adenosine receptors suggested the presence of the A2 adenosine receptor subtype(s) on the cell surface. Recently, we confirmed A2BR expression in native, nontransformed parietal cells at rest by using flow cytometry and confocal microscopy. In this study, we show that A2BR is functional in primary rabbit gastric parietal cells, as indicated by the fact that agonist binding to A2BR increased adenylate cyclase activity and acid production. In addition, both acid production and radioligand binding of adenosine analogs to isolated cell membranes were potently blocked by selective A2BR antagonists, whereas ligands for A1, A2A, and A3 adenosine receptors failed to abolish activation. We conclude that rabbit gastric parietal cells possess functional A2BR proteins that are coupled to Gs and stimulate HCl production upon activation. Whether adenosine- and A2BR-mediated functional responses play a role in human gastric pathophysiology is yet to be elucidated.