Phosphate-buffered saline, solution preparation stock

Washing solution phosphate-buffered saline (PBS). Prepare 10 X stock with 1.37 M NaCl, 27 mM KCl, 100 mM Na2HP04, 18 mM KH2P04 (adjust to pH 7.4 with HCl if necessary) and autoclave before storage at room temperature. Prepare working solution by dilution of one part with nine parts water. 

A Working Solution of the stain should be prepared just before use, by combining 10 ml of 0.1% Triton X-100 in phosphate-buffered saline, 2 mg RNase (DNase-free, Sigma), and 200 pi PI solution (Sigma, 1 mg/ml in distilled water). Stock solutions of Triton X-100 and PI should be stored at 4°C. Centrifuge the ethanol-fixed cells (200 x g for 5 min) and discard the supernatant, removing it completely. 

Prepare an MTT stock solution of 5 mg mH (Sigma, St Louis) in phosphate-buffered saline (PBS), pH 7.5, and filter through a 0.22- x filter to sterilize and remove the small amount of insoluble residue. 

GSH stock solution 32.26 mg (Sigma) dissolved in 25 mL of phosphate buffered saline (PBS Dulbecco s ICN Biomedicals). Prepare fresh on day of assay. 

A 10 mmol L stock coelenterazine solution was prepared by dissolving coelenterazine (Nanolight Technology, Prolume Ltd. Pinetop, AZ, USA) in methanol for use at a final concentration of 10 /tmol L". All coelenterazine solutions were stored at -20 °C and working solutions were kept on ice in the dark during preparation. Diluent buffers comprised distilled water (dH20), Phosphate buffered saline (PBS), Buffer A (10 mmol L Tris [pH 7.8], 1 mmol L EDTA, 0.6 mol L NaCl), 7H9 medium supplemented with Tween-80 with or without 10% OADC (oleic acid, albumin, dextrose, catalase), Luria-Bertani (LB) broth with or... 

Phosphate-buffered saline (PBS) at pH 7.4 was prepared from a stock concentrate. Following dilution, the solution was placed in a vacuum flask and degassed for 1 h. The solution was retained under vacuum and used subsequently for all solution preparations and wash steps. Crystalline protein was weighed and placed in a dry 500-mL llask, and degassed PBS was transferred to the flask to make the following concentrations 2500 mg/dL albumin and 300 mg/dL Cohn I and II fractions. The flask was swirled gently and allowed to sit until all proteins had dissolved (30-40 min). Other solutions were prepared from the stock solutions by serial dilution under vacuum. The stock was discarded after 48 h. 

ABTS radical anions were used according to the method of (Kim et al., 2003). In brief, 1.0 mM of 2, 2 -azobis (2-amidino-propane) dihydrochloride (AAPH), a radical initiator, was mixed with 2.5 mM ABTS in phosphate-buffered saline (pH 7.4) and the mixed solution was heated in a water bath at 68 °C for 13 min. The resulting blue-green ABTS solution was adjusted to the absorbance of 0.650 + 0.020 at 734 nm with additional phosphate-buffered saline. 20 il of sample were added to 980 (iL of the ABTS radical solution. The mixture incubated in a 37°C water bath under restricted light for 10 min. A control (20 iL 50% methanol and 980 mL of ABTS radical solution) was run with each series of samples. The decrease of the absorbance at 734 nm was measured (Cary 50 Scan UV-Visible apparatus) at an endpoint after 10 min. Total antioxidant capacity of plant parts is expressed as mg / g of dry weight of vitamin C equivalents (VCEAC). The radical stock solution had to be freshly prepared and all measurements of the tested samples were repeated at least three times. 

The volume of protein extract solution was adjusted to 5.0 ml with a phosphate reaction buffer consisting of 20 mM phosphates at pH 7.4 and then incubated with 5.0 mg ultra clean carbon/cellobiose core (0.5 ml of stock solution) at 4o C for 24 hours. The final colloid consisted of carbon ceramic particulates coated with a layer of cellobiose and a more superficial layer of adsorbed HIV proteins. These colloidal ceramic viral decoys were prepared for use by clearing unadsorbed material by ultrafiltration dialysis with a stir cell [Filtron, Northborough, MA] mounted with a 100 kd filter and flushed with 100 ml of sterile phosphate buffered saline (injection grade) at 4°C under a N2 pressure head of 10 psi. 

Affinity purified antibodies to E. coli or Salmonella (KPL, Inc., Gaithersburg, MD) were immobilized on the surface of Nylon membranes (courtesy of Pall Corporation, NY) as follows. The Nylon membranes were cut into 1 cm discs and placed into separate wells of a polystyrene plate. Each membrane was incubated (while shaking) for 1 hour in 1 mL of 10% (w/v) caibodiimide solution (pH 5.0). The membranes were washed 3 times (3 minutes each) in 20 mM phosphate buffer solution (pH 5.6). The membranes were transferred to a new polyst)n ene plate and left 3 minutes to dry. Then, 20 pi stock solution (1 mg/ml) of anti-. coli or m -Salmonella antibodies were dropped onto each membrane and left to dry. Following that, each membrane was incubated for 2 hours with 2 ml of 0.5% BSA solution in 20 mM phosphate buffer, pH 5.6. The membranes were then washed 3 times as described above, incorporated into the disposable immunofiltration column and stored at 4 °C until further use. Mortalized bacteria were used in this study for safety reasons and to ensure a static (non-proliferating) sample population for quantitative purposes. Reference quantification of mortaliz bacteria samples were performed by hemacytometer counts and used to prepare stock bacterial suspensions in phosphate-buffered saline. 

The enzyme assay was prepared by mixing 500 yL of 0.100 M phosphate buffer (pH 7.20), 50 yL of ethylhomocholine, 25 yL of 500 mM acetylcholine, and 25 fiL of serum diluted 1 4 with isotonic saline. Stock solutions of the substrate and internal standard were prepared in 10 mM acetate buffer (pH 4.5). The assay proceeded for 10 minutes ambient temperature (22°C) before being stopped by the addition of 25 yL of 2.50 M perchloric acid. This amount of perchloric acid did not stop the reaction instantaneously, but a correction can be made by running a zero-time control. The samples were filtered through 0.45 /urn filters before injection of 20 yL aliquots into the HPLC. 

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