Krebs Ringer buffer

Resuspend the cells at a final density of approx 2 x 106 cells/mL in Krebs-Ringer Buffer containing BSA (1 mg/mL) and fura-2 (2 id. /ml.). 

Resuspend in a suitable volume of Krebs-Ringer buffer to give a final cell density of 2 x 106 cells/mL. To keep this cell density with larger volume cuvets it is recommended to increase the number of cell flasks at the beginning of the experiment. 

Add 100 pL of grannlocytes (10 cells/mL Krebs Ringer s phosphate buffer) to a 6-mL polypropylene tube. 

On day zero,.mice weighing approximately 20 g were inoculated with 5 X 10 Ehrlich ascites tumor cells (EATC) SEM = standard error of the mean. All test solutions were prepared in Krebs Ringers Phosphate buffer and administered by i.p. injection on days 0, 1, 3, 5, 7, 9, 12, 15, and 18. 

The jejunal lumen of rat was perfused in Krebs-Ringer phosphate buffer (pH7.4) containing ImM glucose in the presence or absence of the non-sugar fraction or BS-1. 

Krebs-Ringer/HEPES buffer (KRH) without added calcium NaCI 125 mM KCI 5 mM MgS04 2 mM KH2PO4 1.2 mM HEPES 25 mM EGTA 0.4 mM adjust the pH to 7.4 with NaOH. Store in 20 ml aliquots at -20°C and warm to 37°C before use. This buffer is used to prewash the cells. The actual concentration of free calcium is about 10 [xM. 

Remove medium from cells, and resuspend them in Krebs-Ringer-HEPES and wash them twice with Krebs-Ringer-HEPES buffer by centrifugation. 

Uptake of glutamate results in an acidification of the intravesicular lumen. This acidification can be indicated by the fluorescent dye acridine orange, which accumulates in acid compartments. Synapto-somes were prepared (McMahon etal., 1992) and stored as pellets of about 1 mg overlaid with 200 p.1 Krebs-Ringer-HEPES buffer on ice. Acidification was performed with either glutamate or chloride (Hell et al., 1992 Hartinger and Jahn, 1993). To get rid of the non-vesicular glutamate extensive washing of the permeabilized synaptosomes is required. 

I. Gravimetric Assay. In vitro methods are more sensitive than in vivo methods, but the dose response curves are not as steep, and thus they are less precise. Baake et al. (B2) measured the weight of beef thyroid slices incubated for 24 hours in Krebs-Ringer phosphate buffer. 

The supernatant phase centrifuged at 5,000 x g for 45 minutes was further centrifuged at 24,000 x g for 10 minutes at 4° C The precipitation was removed and the supernatant phase was again centrifuged at 54,000 x g for 60 minutes at 4° C to obtain microsomal fractions. The isolated microsomal fractions were washed twice with Krebs-Ringer phosphate butfer (pH 7.4). Mitochondria and micro ome.s were suspended in Krebs Ringer phosphate buffer (pH 7.4). The final protein concentrations of the mitochondrial and microsomal suspensions were adjusted to 10 mg protein/ml. 

COS cells transfected with pcDNA3.1/V7-3 and the vector pCDNA3.1 served as positive and negative controls, respectively. Parallel incubations with 30 pM of unlabeled anandamide allowed estimation of nonspecific uptake and binding. Uptake assays were carried out for 5 min at 3TC, followed by two washes each with 2 mL of Krebs-Ringer-Henseleit buffer. 

Hanks balanced salt solution (HBSS) [33], developed in 1949, was again a medium free of any Tris of Hepes, but quite deficient in its HCOb concentration (4.2 mM) and Ga/P molar ratio (1.6) in comparison to those of blood. Tyrode solution, with a Ga/P molar ratio of 4.5 and HGOa" concentration of 12 mM, is a Tris- or Hepes-free basal salt medium [34]. Krebs-Ringer bicarbonate buffer (KRBB) [35], developed In 1932, Is a Tris/Hepes-free solution which raises the HGOa concentration of the Ringer solution [36] from zero to 27 mM, i.e., to that of blood plasma. 

Fig 1 shows the effect of the addition of ethanol on the production of uric acid and allantoin by the isolated liver of a fed rat, perfused with a recirculating Krebs-Ringer bicarbonate buffer containing 10 inM glucose Before the addition of ethanol there was a gradual increase in the concentration of uric acid, until a steady state was obtained. The production of allantoin was linear in function of time and reached about 12 nmol/min/g of tissue The addition of 20 mM ethanol provoked a brisk elevation of the concentration of uric acid, which increased to a new steady state This was followed by an approx 2.5-fold enhancement in the rate of production of allantoin. 

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