Isolation of rat lung mast cells for purposes of one-week cultivation using novel Percoll variant Percoll PLUS

Isolation of rat lung mast cells for purposes of one-week cultivation using novel Percoll variant Percoll PLUS

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 separated using a protocol including an improved proteolytic extraction and two...

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Bibliographic Details
Published in:Physiological research Vol. 60; no. 1; pp. 83 - 93
Main Authors: Kubrycht, J, Maxová, H, Nyč, O, Vajner, L, Novotná, J, Hezinová, A, Trnková, A, Vrablová, K, Vytášek, R, Valoušková, V
Format: Journal Article
Language:English
Published: Czech Republic Institute of Physiology 01-01-2011
Subjects:
Animals
Antibiotics
Cell Separation - methods
Centrifugation, Density Gradient
Experiments
Hypoxia
Isotonic Solutions - chemistry
Lung - cytology
Lung - metabolism
Male
Mast Cells - cytology
Povidone - chemistry
Pulmonary arteries
Rats
Silicon Dioxide - chemistry
Studies
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Summary: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 separated using a protocol including an improved proteolytic extraction and two subsequent density gradient separations on Ficoll-Paque 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, interleukins IL-3 and IL-6, and one of two alternative mixtures of antibiotics. These cultivations were performed without any contamination 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-lasting cultivation and observed some model morphological changes of LMC.
ISSN:0862-8408
1802-9973
DOI:10.33549/physiolres.931956