MATERIALS AND METHODS Bacterial strains and culture conditions Two S. aureus strains were used in the present study; S. aureus 8325-4 (SigB-) and SH1000 representing a SigB+.strain. Overnight cultures were grown in Luria Broth (LB) at 37°Cwith shaking at 150 rpm. Exposure to antibiotics was carried out as detailed below. Antibiotics Ciprofloxacin were purchased from Sigma-Aldrich CO. 10 mg/ml stock solution of antibiotic were prepared freshly with 0.1N HCl and stored at -20°C. During the experiment we diluted with sterile water 1:10 and 1:100 depending on the different drug concentration.
Metal chelating activity Briefly, 2 mM FeCl2 was added to different concentrations of test sample and reaction was initiated by the addition of 5 mM ferrozine. The mixture was vigorously shaken and left to stand at room temperature for 10 min. Absorbance was measured at 562 nm after 10 min.8 % Inhibition = [(AB - AA)/AB] x 100, where AB, absorption of blank sample, AA, absorption of test sample. 2.6. Antibacterial
Apparatus- Chromatography column: C18 (10 microns particle size), with Guard column Flow rate: 1.2ml/min Pressure: 30-40kgf Wavelength: 326nm Mobile phase: methanol : water (95:5 v/v) Internal standard: retinyl acetate Injection volume: 20µl Procedure for Retinol extraction from serum samples- 1) 100 µl of serum sample and 100 µl of Retinyl acetate were added into 12 X 100mm glass test tubes. Vortex-mixed for 30 seconds. Then, kept them at 4 C for 5 mins. 2) 1mL of hexane was added and vortex-mixed intermittently for 60 sec. 3) Centrifuged at 2500 rpm for 12 mins.
Serial dilutions were made by sequentially adding 1 ml from 10-1 till 10-7. The serially diluted suspensions (10-1 to10-7) were spread plated on nutrient agar and incubated at 37 ºC for 24 h. The isolated colonies with distinct colony morphology were selected, further streaked to get pure cultures and stored in glycerol at -20oC. All the discrete isolated strains were tested for their plant
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
Subsequently extracted by Microwave at power level 70 for 16 minutes and then filtered. The filtrate obtained, was added 10% HCl (until pH 2-3). Then do the bleaching with NaOCl diluted with water 1: 1 to white. Then converted to sodium alginate by adding 20 g of Na2CO3 and stirred in a mixer. The resulting solution is then etched with ethanol to form sodium alginate fibers.
Then the blower was turned on for sufficient duration and UV lights were switched on for one hour. LAF microbial contamination test (Settling plate method) A total of three petri dishes were prepared aseptically inside a laminar air flow (LAF), and then the petri dish was filled by pouring sterile Tryptone Soy Agar (TSA) liquid media and allowed to solidify. One test media was placed in the LAF cabinet, one test media is placed beside the LAF cabinet while the other the test media was placed near the door. The three petri dishes lid were opened and allowed to stand for 15 minutes and closed again. Then, the test media is then incubated at 37 ° C, for 18-24 hours.
The end product was passed via sieve (no. 85) and stored in desiccators until use. 2.3. Preparation of DS loaded mucoadhesive beads PC-SA [F0], DS-SA [F1] and DS-PC-SA [F2-F6] beads were prepared by the ionotropic gelation method and the compositions are summarized in Table 1. Initially, PC gum was dissolved in distilled water and boiled for 10 min, cooled and stirred for 24 h at
The mixture was incubated for three min at 37ºC in water bath. The clotting time was measured by adding 0.1 ml of CaCl2 to each tube. Three types of sample were used in the FVIII assay; lysate and non lysate of FVIII-loaded RBCs in 1, 1.5, 1.10, and 1.50 dilutions and non-loaded red cells as control sample. Lysate and intact unloaded RBCs were used as a negative
The phosphoamino acids were eluted with water and dried in vacuo. The residues were dissolved in 30l of a mixture of nonradioactive O-phosphoserine and O-Phosphothreonine ( 5 mg of each /ml ), and 10 l aliquots were used for electrophoresis. The electrophoresis buffer contained 25 ml of 88% of formic acid and 87 ml of glacial acetic acid per litre, pH 1.9. The samples were applied to Whatman No.3 MM paper and electrophoresis was carried for 2 hr and 500 V. A small strip of the paper was stained with phosphate reagent. The rest of the strip was stained with 0.2% ninhydrin in acetone.