Isolation of Clara cells from the mouse lung.
A method is described for isolating Clara cells from the mouse lung that does not require the technique of elutriation. Mouse lungs totally perfused of blood are instilled with crystalline trypsin (0.25%) and incubated for the optimum time of 15 min. The lung tissue is chopped, mechanically agitated, and sequentially filtered to obtain a primary digest of 3 to 5 x 10(6) cells. Clara cells, identified routinely by histochemical localization of NADPH diaphorase, using the stain nitrotetrazolium blue (NBT), accounts for between 20 to 40% of the cells in the primary digest. Layering the cells of the primary digest on a discontinuous Percoll gradient followed by centrifugation gives rise to a major band of cells, 52% that are Clara cells (0.77 +/- 0.28 x 10(6)/mouse). A second method was devised to purify the Clara cells by simply centrifuging (32g, 6 min, 10 degrees C) the primary digest and discarding the supernatant that contained only a few NBT positive cells. When this process was repeated three times, the final pellet contained 68% Clara cells realizing 0.55 +/- 0.16 x 10(6) cells/mouse. The cells have typical Clara cell morphology as confirmed by electron microscopy and have a high level of P-450 enzymes (7-ethoxycoumarin deethylase and coumarin hydroxylase). Furthermore, the primary digests and the purified isolates contain less than 1% alveolar Type II cells, although such cells, identified by the histochemical localization of alkaline phosphatase, can be obtained by a second, more extensive digestion procedure. The simple procedure described for the isolation of mouse Clara cells could be further advanced if methods could be devised to prevent the loss of NADPH diaphorase activity during enzymatic digestion and cell centrifugation.
Department of Biochemistry, University College Cardiff, Wales, UK.
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Oreffo
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Morgan
R J
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Richards
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology / Q000648:ultrastructure; D008168:Lung / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008861:Microsomes / Q000032:analysis; D009580:Nitroblue Tetrazolium; D010447:Peptide Hydrolases
D009580:Nitroblue Tetrazolium; D008058:Dihydrolipoamide Dehydrogenase; D010447:Peptide Hydrolases