Isolation of Clara Cells from the Mouse Lung

Abstract

A method is described for isolating Clara cells from the mouse lung that does not require the technique ofelutriation. Mouse lungs totally perfused ofblood 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 106 cells. Clara cells, identified routinely by histochemical localization ofNADPH 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 106/mouse). A second method was devised to purify the Clara cells by simply centrifuging (32g, 6 min, 10 OC) 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 106 cells/mouse. The cells have typical Clara cell morphology as confirmed by electron microscopy and have a high level ofP-450 enzymes (7-ethoxycoumarin deethylase and coumarin hydroxylase). Furthermore, the primary digests and the purified isolates contain less than 1% alveolar lype 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 ofmouse 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.

Molecular organization, expression and chromosomal localization of the mouse pronapsin gene

“…Alveolar macrophages and Clara cells were isolated from the lungs of male CD1 mice and maintained in primary culture as described previously [18]. The cells were consistently isolated with purity in excess of 99%.…”

Polarized Airway Epithelial Models for Immunological Co-Culture Studies

“…However, it is also important to emphasize that primary epithelial cells represent the native and original cells of the microenvironment, which is intended for study, and in theory the optimal candidate for establishing epithelial co-culture models. Similarly, primary bronchial epithelial cells from laboratory animals have been in use over the last 30 years [116,117,118,119,120,121]. Human primary AECs can be obtained in several different ways: (a) autopsy of non-cancerous individuals, (b) atraumatic methods or (c) traumatic methods [121].…”

Micronucleus assay for mouse alveolar Type II and Clara cells

“…Ten unexposed mice were included as controls. 72 hr after the end of the exposure, alveolar Type II cells and Clara cells were extracted from the lungs using methods previously developed for rats and mice [Oreffo et al, 1990;De Boeck et al, 2003] with some modifications as described later. Two methods for cell isolation were used.…”

“…The development of isolation methods for alveolar Type II cells and Clara cells in rodents started about two decades ago [Devereux and Fouts, 1980;Dobbs et al, 1986Dobbs et al, , 1990Oreffo et al, 1990], and many improvements to enhance the purity of the isolated cells, especially concerning alveolar Type II cells, have been subsequently suggested [Dobbs et al, 1986;Chen et al, 2004]. Dobbs et al [1986] described a method to isolate alveolar Type II cells in rats using elastase instead of trypsin to digest the lung tissue.…”

“…In this study, our objective was to develop a relatively simple MN assay to detect genotoxic damage after inhalation exposure in mouse alveolar Type II and Clara cells, which are potential target cells for lung carcinogens. The isolation techniques we used are based on previously published methods [Oreffo et al, 1990;De Boeck et al, 2003]. As a model carcinogen we used ethylene oxide, a direct-acting alkylating agent which has been shown to induce MNi in various in vivo and in vitro assays [Zhong et al, 1991;Vergnes and Pritts, 1994;Lorenti Garcia et al, 2001].…”