Fetal Bovine Serum (FBS) in 10 %, DMSO and 96 well plate microplate reader were the other materials of the experiment. 1.2 METHODS OF MTT ASSAY The HEK-293 cells were seed into culture medium which was 100 μl. After that, cells were incubated at 37oC and CO2 with 5% during 24 hours. At the end of 24 hours, the plasmid with Pin1 gene was transfected to HEK-293 by the help of PEI reagent and cationic lipid method. Plasmid and PEI reagent were prepared both 1:3 and 1:5
Animals from week 8 to week 16 of age were prepared for standard renal clearance by housing in metabolic cages for four days to asses urine volume, glomerular filtration rate (GFR) and sodium excretion. Expressions of renal sodium transporters including NHE-3, NKCC2, α- ENaC and α1- Na+: K+ ATPase, were determined in 16- week old male rats at mRNA and protein levels. At the end of the experiment, animals anaesthetized by Inactin and the efficiency of the renal clearance was tested after challenge with hypertonic saline ( 5 % Na Cl )
Air-conditioned rooms will be used by the animal holding facility and the test animals will be placed under 12 hours dark/12 hours light condition wherein the area will be lighted from 6:00AM to 6:00PM and keeping the lights off for the next 12 hours. The rats will be acclimatized for one (1) week before the start of the biological tests. Every other day, the cages will be cleaned properly dried and disinfected. The rats will be fed with commercial rat pellets and will be given sufficient water. Crushed rabbit pellets and corn kernel can be an alternative food.
All the samples were collected in a sterile container and transferred to the laboratory. One gram of soil sample was suspended in 10ml of distilled water and one ml of curd was taken and these samples were mixed thoroughly (gravity cream was removed from curd samples as it may cause contamination of other microbes) and emulsification of the samples was done using 10ml of 1% sodium carbonate solution (freshly prepared) and was kept at 37ºC for 24 hours for incubation. This prevents the growth of gram-negative organisms (Dundar et al., 2003). These samples were then subjected to serial dilution up to 10-6 dilutions. From dilutions 10-4 to 10-6 , 0.1 ml of each sample was spread onto nutrient agar plates containing 0.0016% (w/v) sodium azide and were kept for incubation at 37ºC for 24-48 hrs.
After oral administration, the mice were observed on an hourly basis for 24 h to assess the general behavior, any signs of toxicity, and mortality. They were further observed for 14 days for toxic symptoms and mortality. Mice were weighed before starting and ending acute toxicity and any changes in skin fur, eyes, mucous membranes, central nervous system and behavioral pattern were observed. Tremors, convulsions, salivation, diarrhea, lethargy, sleep, coma and any specific severe sign were also
Each tube was then dragged into the spectrophotometer to be analyzed. A data point for each analyzed tube was placed on the graph to show the optical density and glucose concentrations. After graphing this data, part two needed to be completed. To start, 5 different test tubes were filled with 3 drops of 5 different patients blood followed by 5 drops of deionized water. Next, 5 drops of Barium Hydroxide was placed in each tube to clear proteins and cell membranes for an accurate reading could be made.
Group2: Administered garlic ethanolic extract for fourteen days to diabetic rats. Group3: Glibenclamide administered 600ug/kg body weight for fourteen days. Group4: Untreated group. Biochemical assay: Take blood from rat heart and check the concentration of insulin serum glucose. We check insulin by using radio immune assay kit.
(normal is 60-125 mg/dL), glucose in the urine, as well as a bladder infection. With the information gained from the physical exam and the lab results Sasha was diagnosed with diabetes. We spoke in depth about diabetes with the owner and counselled the owner about the appropriate care, treatment options, and clinical signs to watch for. We immediately started Sasha on insulin, a special diet low in calories and high in fibre, and antibiotics to treat the bladder infection. Insulin is a medication that is given twice a day under the skin with a special syringe and needle to treat the diabetes.
The amount of formazan crystals formed was measured after 2 hours of exposure to the MTT solution in dimethyl sulfoxide (DMSO) and absorbance values were measured at 540 nm by a scanning multi well spectrophotometer plate reader (ELISA reader). Cytotoxicity experiments were performed in triplicate and results were presented as the mean ± standard deviation. 2.6.3 In vivo study The biocompatibility properties of the presently fabricated substrates were evaluated for an inflammatory host tissue response through in vivo tests. For this, mice were anesthetized by an intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg) and the present samples were implanted into the subcutaneous dorsal area of individual Swiss mice and controls were used (without material) (n=3) for 7 days. The samples were extracted at the end of the time period together with the adherent tissue for histological preparation and examination, and were fixed in a 10% formaldehyde solution for several days, embedded in paraffin, sectioned and stained using the classical hematoxylin–eosin method.
Transect quadrat method Include pictures of the actual quadrats made. 3.4 Counting of Samples In each quadrat, all the bivalves present regardless of its species were counted. The data collected were the number of existing bivalves on each site based from the transect-quadrat line, which was counted manually without age consideration. The bivalves sampled were not collected. Therefore, after the sampling of the bivalves, the organisms were just left on the sampling sites.