Water, acid hypotonic

For potassium depletion, cells are washed with potassium-free buffer (140 mM NaCl, 20 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), ImM CaCh, Img/mL o-glucose, pH 7.4) and then rinsed in hypotonic buffer (potassium-free buffer 1 1 diluted with distilled water) for five minutes. Then, cells are quickly washed three times in potassium-free buffer followed by incubation for 20 minutes at 37°C in buffer. Control experiments are performed in the same manner, except all solutions additionally contain 10 mM KCl. 

Inhalation of distilled water will cause bronchoconstriction in 60% of infants with a history of wheeze (SEDA-17, 212). K nebulizer solutions are hypotonic or hypertonic or contain preservatives (such as benzalkonium chloride, edetic acid, sulfites and metabisulfite) they can cause paradoxical bronchoconstriction. It has been recommended that aU nebulizer solutions should be isotonic and free of preservatives (SEDA-13,132). Unit dose vials are available in which the drug solution is isotonic and preservative-free. However, multidose formulations are stiU available and are widely used, as the cost per dose is less. 

A high speed hybridization procedure (total, including fixation and detection, 1 h) has been described by Bourinbaiar et al. (1991). They suspended washed lymphocytes, for the detection of HIV, in a mildly hypotonic medium (1 part medium and 2 parts distilled water) and spread 25 xl ( 10 cells) in 8 mm wells of Teflon-coated multiwell glass slides and fixed the cells in Carnoy s II solution (60% methanol, 30% chloroform, 10% glacial acetic acid) for 10 min at room temperature and then for 10 min in absolute acetone. Five microliters of biotinylated probes (100 ng/ml) is added to the dried cells and the slides are covered with autoclavable plastic (cut from biohazard bag) and placed in a microwave oven. The position of the slide in the oven and the incubation time are crucial for this hybridization step. In microwave ovens with a carousel, the slide is placed about 6 cm from the center and a 150 ml beaker filled with tap water is placed in the center. The slide is then irradiated for 30 s on Defrost . The probes can be detected after a rinse with 2 x SSC. 

Hypotonic pretreatment of seminiferous tubules can be done with distilled water. The swelling of cells encourages the spreading of chromosomes while preparing the slides. The duration of hypotonicity is varied depending upon the age of the animal. When young animals are to be studied, hypotonic treatment is less than 5 min, whereas seminiferous tubules from adults require at least 10 min of hypotonic treatment. Place hypotonically treated seminiferous tubules in 3 1 fixative (three parts of absolute ethyl alcohol and one part of glacial acetic acid) for 25 min. 

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