Krebs-bicarbonate buffer

Fig. 4 Superfusion neurotransmitter release assay in synaptosomes. (a) Schematic drawing of a superfusion setup. Synaptosomes are preloaded with radioactive neurotransmitter and captured on fiberglass filters in superfusion chambers under continuous superfusion with gassed physiological salt solution (e.g., Krebs bicarbonate buffer) using a peristaltic pump. After a 10- to 15-minute wash, neurotransmitter release is triggered by rapid switching of superfusion lines to a stimulating buffer (e.g., high-potassium solution). Superfusate is collected on a fraction collector, and radioactivity is measured by liquid scintillation, (b) Typical trace recording of tritium-labeled norepinephrine fractional release in rat cortical synaptosomes stimulated by high potassium and a-latrotoxin in the presence or absence of external calcium.

In studies performed by Endre et al., rat kidneys were perfused with Rb+-containing Krebs bicarbonate buffer.55,95 A tracer quantity of radioactive 86Rb+ was added to the perfusate in some experiments to evaluate the NMR visibility of the intracellular 87 Rb. The uptake of NMR-detectable Rb+ into the kidney was analysed in terms of the model discussed above. The kinetic parameters obtained from three experiments were V b + kIE = 0.92 0.07 /xmol/ming,... 

Rat Hepatocytes Rat hepatocytes are freshly isolated by perfusion and are diluted with Krebs-bicarbonate buffer (pH 7.4) to 1 x 10 cells/mL. Cell viability is analyzed by the trypan blue exclusion method. 

The GS-AM Standard Curve lAM stock solution (10 mM, 100 xL) in 10 mM ammonium bicarbonate buffer (pH 10) is added to amber centrifuge tubes, to which 100 pL of various GSH stock solutions in Krebs-bicarbonate buffer (pH 7.4) is added. The final GSH concentrations are 1, 5, 10,25, 50, and 100 pM. All samples are vortexed and are placed in dark at room temperature for 60 min. Derivatization reaction is quenched by the addition of 400 pL of acetonitrile. The resulting mixture is mixed with 25 pL of -y-Glu-Glu stock solution (0.25 mM, internal standard) in water/aeetonitrile (1 1). After centrifugation, the supernatant (300 pL) is transferred to a 96-well plate for LC/MS/MS analysis. 

Bally et al. (1960) incubated epididymal adipose tissue from ad libitum fed rats with palmitate- -C in a Krebs bicarbonate buffer containing 3 % human albumin. They found that the addition of small concentrations of glucose increased the incorporation of palmitate into tissue lipid,... 

Epididymal adipose tissue from normal fed rats incubated with glucose-in a Krebs bicarbonate buffer produces significant quantities of C 02... 

The double-isotope dansyl microassay has also been used to measure release of amino acids from brain preparations in vitro (Snodgrass and Iversen, 1973). Rat cortical tissue was chopped using a Mcllwam chopper and suspended in Krebs-bi-carbonate-glucose medium. Electrical stimulation was applied and, following superfusion, the medium was changed for a Krebs-bicarbonate buffer containing 55 mM KCl. In this way,... 

By the late 1920s quantitative micro-determination of oxygen uptake had been developed in Warburg s laboratory in Berlin based on a manometric technique introduced by Barcroft and Haldane (1902). With this equipment evolution of carbon dioxide or uptake of oxygen could be monitored Carbon dioxide produced in respiration was absorbed by potassium hydroxide. If bicarbonate buffer was used, acid production caused carbon dioxide to be released. Krebs and others from... 

After the equilibration period, clearance periods of 20 min are used. Urine samples are collected and perfusate is obtained at midpoint of the clearance period for the evaluation of overall kidney function. For determination of glomerular filtration rate (GFR) and fluid transport, 3H-labelled polyethylene glycol is added to a modified Krebs-Henseleit bicarbonate buffer. Electrolytes are determined in urine by standard flame photometry. Fractional excretions of water, electrolytes and test compounds are calculated. 

Hearts from CD-I male rats (250-300 g n=4 each group, 34 total) are excised and perfused within 25 s to 2 min with non-recirculating oxygenated Krebs-Henseleit bicarbonate buffer (120 mM NaCl, 25 mM NaHCOj, 1.2 mM MgSO -ZHp, 5 mM KCl, 1.7 mM CaCl-2H20, 10 mM glucose, pH 7.4, 37°C) at a constant coronary perfusion pressure (CPP) of 80 mm Hg (18, 19). 

Experiment I. In a time-course experiment, mucosal PGE production and phospholipid fatty acid profile were assessed at d 0,4,8,12, and 16 of dietary treatment in formula-fed and naturally reared piglets (n = 5 piglets per time per dietary treatment). Mucosal cells were scraped from proximal ends of the small intestine and frozen at -80°C for later lipid analysis. Lipids were extracted by a modified Folch procedure (15). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were separated by thin-layer chromatography (16), and fatty acids in each phospholipid fraction were analyzed by gas chromatography. For eicosanoid measures, fresh mucosal tissue was incubated in Kreb s Ringer bicarbonate buffer as described previously (17). PGE2 was extracted from the incubation media with ethyl acetate and quantified using a competitive enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI). 

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. 

Isolated rat liver hepatocytes were prepared as described by Seglen (9), except that the cells were suspended and incubated in Krebs-Henseleit bicarbonate buffer (K-H buffer), supplemented as specified. 

Our approach to this subject was to develop an to vitro model system in which we could study the effects of tropic hormones upon steroidogenesis to the corpus luteum under precisely controlled conditions. Essentially the system we worked out was to prepare slices of a single corpus luteum, obtained from a pregnant cow at slaughter, and to incubate these slices in Krebs-Ringer bicarbonate buffer in the presence or absence of test substances (3). After the incubation (usually 2 hours) progesterone was isolated from the tissue and medium, and measured spectrophotometrically. 

The height of each bar represents the mean and tiie brackets the 95% confidence limit. Each vessel contained 5 ml Krebs-Rir er bicarbonate buffer and approximately 0,3 to 0.5 g slices, The slices were incubated for 1 hour in buffer alone, and then transferred to fresh buffer alone or buffer containing 10 jug LH/5 ml for the second incubation. After 15 minutes, one set of slices was analyzed for cyclic AMP, The other set of slices was incubated for 2 hours and steroid synthesis measured,... 

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