Comparison of enzyme-linked immunosorbent assay and Fassisi® bovine immunoglobulin G (IgG) immunoassay for quantification of bovine IgG in neonatal calf serum

Background and Aim: Rapid tests are routinely used to estimate serum immunoglobulin G (IgG) concentrations in diagnosing a failure of passive transfer (FPT) in calves. The study aimed to compare the Fassisi® Bovine IgG (FB-IgG) immunoassay and an enzyme-linked immunosorbent assay for quantifying bovine IgG in neonatal calf serum. Materials and Methods: A total of 277 calves of 1-10 days of age were used in this study. Blood samples were obtained, and serum was extracted by centrifuging the samples at 2740× g for 5 min at 20°C. The serum was analyzed using the FB-IgG according to the manufacturer's specifications. Serum IgG concentrations were also determined by enzyme-linked immunosorbent assay (ELISA-IgG). FPT was defined as a serum IgG concentration <10 mg/mL. Results: The mean ELISA-IgG serum concentration was 8.40 mg/mL (SD=7.02, range=0.10-47.50 mg/mL). FPT prevalence based on the ELISA measurements was 66.8%. The prevalence of partial and full FPT based on the FB-IgG was 54.5%. The ELISA-IgG and FB-IgG results were subjected to correlation and regression analysis. Overall sensitivity and specificity of the FB-IgG were 61.1% and 58.7%, respectively. A statistically significant dependence on age was identified in the results. Conclusion: Our findings suggest that the FB-IgG rapid method is less accurate and provides no other advantages over established methods.

Effects of Epidermal Growth Factor and Myo-Inositol on Nuclear Configuration and Subsequent Embryonic Development of Sheep Oocytes

Growth factors and vitamin-like substances have great positive importance in most biological interactions in the cellular level. The addition of these elements in the culture media will increase the yield of the resulting embryos and improve quality. We examined the effects of epidermal growth factor (EGF) and myo-inositol (MI) on meiotic maturation and yields of blastocyst of Awassi sheep oocyte across two experiments. The oocytes obtained were subjected into three treatments: A (without EGF nor MI), B (10 ng/ml EGF + 20 mmol/l MI) and C (50 ng/ml EGF +40 mmol/l MI). Oocytes were then cultured in Ham's F-10 medium supplemented with 5% (v: v) fetal calf serum and 40 ng/ml follicle - stimulating hormone. In the first experiment, during the 27-h culture period, the oocytes were assessed for germinal vesicle break down, metaphase-I and metaphase-II stages across three-time intervals (9, 21 and 27-h). Results of the experiment showed that EGF and MI enhanced the rates of germinal vesicle break down phase (1.53%; 27-h interval; lowest value), metaphase-I (33.87%; 21-h interval) and metaphase-II (89.23%; 27-h interval). In the second experiment, the oocytes incubated in treatment B achieved the highest rates of cleavage (81.96%), 2-8 cell (62.35%) and blastocyst (45.09%). It is concluded from the present study that incubating sheep oocytes in culture media containing a cocktail of EGF (10 ng/ml) and MI (20 mmol/l) significantly improves the rates of metaphase-II, fertilization and blastocyst rates.

New Born Calf Serum Can Induce Spheroid Formation in Breast Cancer KAIMRC1 Cell Line

Three-dimensional (3D) cell culture systems have become very popular in the field of drug screening and discovery. There is an immense demand for highly efficient and easy methods to produce 3D spheroids in any cell format. We have developed a novel and easy method to produce spheroids from the newly isolated KAIMRC1 cell line in vitro. It can be used as a 3D model to study proliferation, differentiation, cell death, and drug response of cancer cells. Our procedure requires growth media supplemented with 10% new born calf serum (NBCS) and regular cell culture plates to generate KAIMRC1 spheroids without the need for any specialized 3D cell culture system. This procedure generates multiple spheroids within a 12–24-h culture. KAIMRC1 spheroids are compact, homogeneous in size and morphology with a mean size of 55.8 µm (±3.5). High content imaging (HCI) of KAIMRC1 spheroids treated with a panel of 240 compounds resulted in the identification of several highly specific compounds towards spheroids. Immunophenotyping of KAIMRC1 spheroids revealed phosphorylation of FAK, cJUN, and E-cadherin, which suggests the involvement of JNK/JUN pathway in the KAIMRC1 spheroids formation. Gene expression analysis showed upregulation of cell junction genes, GJB3, DSC1, CLDN5, CLDN8, and PLAU. Furthermore, co-culture of KAIMRC1 cells with primary cancer-associated-fibroblasts (CAFs) showcased the potential of these cells in drug discovery application.

Lateral Flow Immunoassay with Quantum-Dot-Embedded Silica Nanoparticles for Prostate-Specific Antigen Detection

Prostate cancer can be detected early by testing the presence of prostate-specific antigen (PSA) in the blood. Lateral flow immunoassay (LFIA) has been used because it is cost effective and easy to use and also has a rapid sample-to-answer process. Quantum dots (QDs) with very bright fluorescence have been previously used to improve the detection sensitivity of LFIAs. In the current study, a highly sensitive LFIA kit was devised using QD-embedded silica nanoparticles. In the present study, only a smartphone and a computer software program, ImageJ, were used, because the developed system had high sensitivity by using very bright nanoprobes. The limit of PSA detection of the developed LFIA system was 0.138 ng/mL. The area under the curve of this system was calculated as 0.852. The system did not show any false-negative result when 47 human serum samples were analyzed; it only detected PSA and did not detect alpha-fetoprotein and newborn calf serum in the samples. Additionally, fluorescence was maintained on the strip for 10 d after the test. With its high sensitivity and convenience, the devised LFIA kit can be used for the diagnosis of prostate cancer.

In Vitro Effects of Natrum muriaticum in Kidney Cell Lines MDCK and LLC-PK1

Previous papers have indicated that homeopathic solutions modify the cellular and biochemical aspects of cells maintained in culture. In this study, the effects of Natrum muriaticum, a medicine used in the homeopathic clinic for the treatment of hypertension, were evaluated in kidney MDCK and LLC-PK1 cell lines. The following cellular parameters were analyzed: viability, morphology and expression of the (Na+-K+)-ATPase and the angiotensin II receptors AT1 and AT2. The cell lines were plated (5.0 x 104 cells/mL) in DMEM supplemented with 10% fetal calf serum (FCS). After 24 hours at 37°C, DMEM was re-fed with the addition of 10% (V/V) and 1% (V/V) of the following samples: Natrum muriaticum 30CH, water 30CH and non-dynamized sterile distilled water to do the MTT assay. The results obtained from these groups were compared to those obtained by incubation of the cells in culture medium free of these solutions (Control). Cell viability was assessed by a colorimetric MTT ELISA assay (490nm). The values from four independent experiments performed in quintuplicate were plotted and statistically analyzed by Sigma Plot v.11 (Jandel Scientific). The morphology of MDCK cells was evaluated by optical microscope after Giemsa’s staining. The expression of the (Na++K+)-ATPase and AT1/AT2 of LLC-PK1 cells was evaluated by Western Blot (WB) analysis. For this experimental set, 5.0x104 cells/mL were incubated in DMEM supplemented with 10% FCS and daily culture medium was replaced by a new one, containing: Natrum muriaticum 30CH and water 30CH. Additionally, cells were treated for 5, 10 and 15 days with 1% of specific solutions and the total protein was measured by the Lowry method, after cell lysis. The samples were analyzed by electrophoresis in SDS-PAGE (12% gel) and transferred to nitrocellulose membrane. This membrane was incubated with specific primary antibodies (anti-(Na++K+)-ATPase, anti-AT1 and AT2 or anti-anti-beta-actin). The detection was performed using ECL system and Hyperfilm. MTT assays showed a statistically significant reduction in cellular mitochondrial activity (p

Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro

Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na–citrate tubes revealed the highest count of vital cells compared to commercial Li–heparin (p < 0.01) and K3EDTA (p < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced (p < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) (p < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.

The reaggregation of normal granulosa-cumulus cells and mouse oocytes with polycystic ovarian syndrome in vitro: An experimental study

Background: The dialogue between oocytes and their surrounding cells plays a major role in the progress of oocyte meiosis and their developmental potential. Objective: This study aimed to evaluate the effect of co-culture of normal granulosa-cumulus cells (GCCs) with oocytes from polycystic ovarian syndrome (PCOS) mice. Materials and Methods: Normal GCCs were collected from 10 virgin adult Naval Medical Research Institute female mice (30-35 gr, 7-8 wk old), and were cultured in an alpha-minimum essential medium supplemented with 5% fetal calf serum for 24-48 hr (1×106 cells/well). Then, germinal-vesicle oocytes from PCOS mice were cultured in the presence of cultured normal GCCs (experimental group) and without GCCs (control group). The maturation rate and quality of the PCOS oocytes were examined by evaluating TFAM and Cx43 gene expression (real-time PCR) and the connection among PCOS oocytes and normal GCCs after 24 hr of culture. Results: The co-culture of normal GCCs and PCOS oocytes in the experimental group led to the formation of a complex called a PCOS oocyte-normal GCCs complex. The maturation rate of these complexes was significantly increased compared to that of the control group (p ≤ 0.001). A significant difference was also found in the expression of Cx43 (p ≤ 0.001) and TFAM (p < 0.05) genes in the experimental group compared with the control group. The connection between PCOS oocytes and normal GCCs was observed in the scanning electron microscope images. Conclusion: Co-culture with normal GCCs improves the capacity of PCOS oocytes to enter meiosis, which may result in the promotion of assisted reproduction techniques. Key words: PCOS, Co-culture, Granulosa-cumulus cells, IVM, Cx43.

Tumor-Derived Cell Culture Model for the Investigation of Meningioma Biology

Meningioma is the most common primary central nervous system tumor. Although mostly nonmalignant, meningioma can cause serious complications by mass effect and vasogenic edema. While surgery and radiation improve outcomes, not all cases can be treated due to eloquent location. Presently no medical treatment is available to slow meningioma growth owing to incomplete understanding of the underlying pathology, which in turn is due to the lack of high-fidelity tissue culture and animal models. We propose a simple and rapid method for the establishment of meningioma tumor-derived primary cultures. These cells can be maintained in culture for a limited time in serum-free media as spheres and form adherent cultures in the presence of 4% fetal calf serum. Many of the tissue samples show expression of the lineage marker PDG2S, which is typically retained in matched cultured cells, suggesting the presence of cells of arachnoid origin. Furthermore, nonarachnoid cells including vascular endothelial cells are also present in the cultures in addition to arachnoid cells, potentially providing a more accurate tumor cell microenvironment, and thus making the model more relevant for meningioma research and high-throughput drug screening.

Proliferation and Differentiation of Intestinal Caco-2 Cells Are Maintained in Culture with Human Platelet Lysate Instead of Fetal Calf Serum

Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces a high variability, risk of contamination and is ethically disputed. We tested the culture of Caco-2 cells with human platelet lysate (PL) instead of FCS. We compared cell viability and differentiation by measuring ATP levels, gene and protein expression of specific markers in total cell extracts, brush border membrane vesicles (BBM) and lipid rafts (LR). Cell viability was slightly enhanced in cells grown with PL compared to FCS. The cells differentiated to an intestinal phenotype like the cells cultured in FCS, as indicated by the similar gene expression levels of hexose and protein transport proteins and the structural protein VILLIN. BBM showed a comparable distribution of the intestinal hydrolases, indicating a maintained cell membrane polarity. The distribution of the marker protein FLOTILLIN-2 in LR was also similar. We conclude that PL is an exquisite and suitable replacement for FCS in the culture of Caco-2 cells that can eliminate many disadvantages incurred due to the use of FCS.