Prolonged cultivation of separated rat lung mast cells (LMC) in vitro is necessary to better investigate a possible role of LMC in different stages of tissue remodeling induced by hypoxia. Rat lung mast cells (LMC) were sepa rated using a protocol including an improved proteolytic extracti on and two subsequent density gradient separations on Ficoll-P aque PLUS and a new generation of Percoll, i.e. Percoll PLUS. Instead of usual isotonic stock Percoll solution, an alternative “asymptotically isotonic” stock solution was more successful in our density separation of LMC on Percoll PLUS. Separated cells were cultivated for six days in media including stem cell factor, interleu kins IL-3 and IL-6, and one of two alternative mixtures of antibi otics. These cultivations were performed without any contaminatio n and with only rare changes in cell size and morphology. Model co-cultivation of two allogenic fractions of LMC often caused considerable rapid changes in cell morphology and size. In contrast to these observations no or rare morphological changes were found after cultivation under hypoxic conditions. In conclusions, we modified separation on Percoll PLUS to be widely used, altered LMC separation with respect to purposes of long-lasti ng cultivation and observed some model morphological changes of LMC., J. Kubrycht ... [et al.]., and Obsahuje bibliografii a bibliografické odkazy
An attempt was made to assess whether the choice of the gradient media could influence the yield of basolateral membrane vesicles isolated from the rat intestine as well as their functional characteristics. Crude membranes prepared in the same way were therefore centrifuged with 10 % Percoll, on a discontinuous sucrose gradient or on a continuous sorbitol gradient. The protein yield was significantly higher with the Percoll gradient than with sucrose and sorbitol gradient centrifugation (2.7 ±1.0 %; 0.4 ±0.1 %; 0.6±0.2 %, respectively). Enrichment in Ma+,K+-ATPase was similar in all three preparations (8.50±2.34; 8.22±4.78; 8.20±2.08). However, contamination with brush border membranes was significantly higher after Percoll gradient centrifugation and negligible after the use of the other two gradient media. Transport of D-glucose in the BLM prepared by Percoll gradient centrifugation also indicated some contamination with functional brush-border membranes. An attempt to purify basolateral membrane vesicles after Percoll gradient centrifugation with Ca2+ precipitation, however, reduced the protein yield to less than 1 %. We conclude that in the preparation of basolateral membrane vesicles from the rat enterocytes each of the gradient media may have certain advantages and disadvantages, which should be considered according to the purpose of the preparation.