Perfusion buffer solution preparation

One approach that has been used quite widely to quantitate neurotransmitter release employs radiolabeled (tritiated) neurotransmitter analogs (e.g.. Reference 67). First, tissue is incubated in a buffer solution that contains tritiated neurotransmitter. During this time, the radiolabeled transmitter is taken up into cells by endogenous plasma-membrane transporters and packaged into vesicles by vesicular transporters. The tissue preparation then is rinsed in buffer to remove extracellular radiolabeled transmitter leaving only that which was taken up into cells. This stored transmitter is then released over time by exocytosis. To quantitate its release, the tissue is continuously perfused with buffer, and time-dependent aliquots are collected. Radioactivity is measured in the aliquots with a scintillation counter and is used as an index of endogenous neurotransmitter release. Rather than estimate absolute neurotransmitter release, this method is typically used to compare the relative release between two or more conditions. 

In another configuration [53] two different approaches for pesticide analysis were employed. A crude enzyme solution capable of hydrolyzing organophos-phate insecticides was prepared. The enzyme was coupled to controlled-pore glass with glutaraldehyde. The insecticides, e.g., parathion, cyanophos, and dia-zinon, were dissolved in a perfusion buffer (Tris pH 8.9,1 % Triton X-100) and injected as a 10 min pulse into an ET in split-flow mode. The instrument measured the heat output due to insecticide hydrolysis and consecutive buffer pro-tonization. For parathion, the detection limit was approximately 10 ppm. 

Sample preparation Urine. Add 10 pL urine diluted 10 times with 10 mM pH 7.5 Na2HP04 buffer to 1-5 pg nizatidine, make up volume to 300 pL with 10 mM pH 7.5 Na2HP04 buffer. Place solution on YM-10 ultrafiltration membrane with a cut-off of 10000, centrifuge at 4000 g for 20 min. Mix 180 pL filtrate with 20 pL MeOH, inject 50 pL. Perfusate. Add 10-100 pL perfusate to 1-5 pg nizatidine, make up volume to 300 pL with 10 mM pH 7.5 Na2HP04 buffer. Place solution on YM-10 ultrafiltration membrane with a cut-off of 10 000, centrifuge at 4000 g for 20 min. Mix 180 pL filtrate with 20 pL MeOH, inject 50 pL. 

Intestinal Perfusion Experiments. Hydrolysate solutions for use in the perfused Intestine experiments were prepared by concentrating the pronase hydrolysates approximately two-fold in a rotary evaporator at 60 C (to inactivate pronase, 24) and mixing the equivalent of 157.5 mg (untreated zeln), 289.2 mg (Ca(0H)2-treated zein) or 150.0 mg (NaOH-treated zein) with 0.05M HEPES (Calbio-chem-Behring Corp., LaJolla, CA) buffer (pH 8.0). The final solutions had a pH of 8.10 0.05. 

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