Isolation of alveolar type II cells by centrifugal elutriation

Summary Centrifugal elutriation (counterflow centrifugation) was used to develop a reproducible method for obtaining a nearly pure population of isolated alveolar type II cells. Lung was dissociated into individual cells with recrystallized trypsin, and the type II cells were partially purified by centrifugation on a discontinuous density gradient. The alveolar type II cells were finally purified by centrifugal elutriation. Cells were collected from the elutriator rotor by stepwise increases in flow rates. Cells obtained at flow rates of 7 and 14 ml per min were lymphocytes, other small cells, a few type II cells and cell debris; cells collected at flow rates of 18 and 22 ml per min were mainly type II cells; and cells collected at flow rates of 28, 34 and 43 ml per min were macrophages, some type II cells, other lung cells and cell aggregates. At flow rates of 18 and 22 ml per min, 1.9±1.0×10⁶ cells per rat lung (mean±S.D.,n=30) were recovered of which 86±6% were type II cells. At these flow rates, 94% of the cells excluded the vital dye erythrosin B from their cytoplasm. They consumed oxygen at a rate of 101±21 nmol per hr·10⁶ cells (mean±S.D.,n=4), and their oxygen consumption increased only 10% after 10mm sodium succinate was added. The cells incorporated [¹⁴C]leucine into protein and lipid for 4 hr. Electron micrographs of the cells collected at flow rates of 18 and 22 ml per min show a high percentage of morphologically intact alveolar type II cells. We conclude that centrifugal elutriation is a reproducible method for obtaining nearly pure, metabolically active alveolar type II cells. Postdoctoral trainee supported by Grants HL-05251 and HL-07192 from the National Heart, Lung and Blood Institute. This work was supported by U.S. Public Health Service Grants Program-Project HL-06285 and Pediatric Pulmonary SCOR HL-19185, and by a grant-in-aid from the American Heart Association (77-1098).     

Cytochrome P-450 monooxygenase,epoxide hydrolase and flavin monooxygenase activities in clara cells and alveolar type II cells isolated from rabbit

The activities of several enzymes which metabolize xenobiotics were measured and compared in freshly isolated rabbit Clara cells (50–70% purity) and alveolar type II cells (80–95% purity) or microsomal preparations from the isolated cell fractions. The presence of 1 mM nicotinamide in protease and cell isolation buffers increased significantly 7-ethoxycoumarin (7-EC) deethylase and epoxide hydrolase activities in the isolated Clara and type II cells. Isolated Clara cell fractions metabolized 7-EC to umbelliferone at a rate of 241 ± 27 pmoles/mg prot/min (mean ± S.E., N=5), while the 7-EC deethylation rate in type II cells was 111 ± 15 pmoles/mg prot/min. Coumarin hydroxylation activity, however, was more than ten times greater in the Clara cells than in the type II cells on a per mg cellular protein basis. N-oxidation of N,N-dimethylaniline, catalyzed by a flavin monooxygenase, was about 2 times as great in microsomes of Clara cells as in microsomes of type II cells. Epoxide hydrolase activity with benzo(a)pyrene 4,5-oxide as substrate was about 10 times higher in Clara cells than in type II cells. Because of the greater cellular, structural and functional heterogeneity in lung, differential distribution of enzymes responsible for xenobiotic metabolism in this tissue may contribute to cell selective chemical toxicity and carcinogenesis.Abbreviations 7-EC 7-ethoxycoumarin - DMA N,N-dimethylaniline     

Theory and practice of centrifugal elutriation (CE)

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that aselective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5×10⁸ rbc) resulted in better separations than low cell loads (5×10⁷ rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.     

Nonselective voltage-gated ionic channels in type II taste cells

In cells of different types outward voltage-gated (VG) ion currents are generally carried by potassium ions. However, in mouse type II taste cells these currents persist when K⁺-selective ion channels are inhibited. In this study, we examined the ion channels that provide a pathway for atypical VG outward currents in type II taste cells. These channels are found to be weakly selective and permeabile to large molecules such as NMDG, gluconate, and ATP. According to non-stationary fluctuation analysis, single channel conductance is about 200 pS. The data obtained suggest that the nonselective ion channels are similar to hemichannels formed by connexins, the gap-junction proteins, in the plasma membrane of vertebrate cells.     

Effect of Escherichia coli lipopolysaccharide on phosphatidylcholine biosynthesis by rat lung and alveolar type II cells

Summary Alterations in pulmonary surfactant are partly responsible for the respiratory insufficiency seen under septic shock process. We have used an experimental model of LPS-induced shock in rats to examine the cells responsible for the pulmonary surfactant synthesis and its relationship to lung injury. (¹⁴C)Choline incorporation into phosphatidylcholine was significantly reduced in lung homogenates or type II cells obtained from LPS-treated animals. Addition of LPS in vitro fails to increase (¹⁴C)choline incorporation in type II cells obtained from LPS-treated animals. We suggest that this depression of pulmonary phosphatidylcholine synthesis may partly explain the occurrence of respiratory failure with septic shock.     

LysoTracker is a marker of differentiated alveolar type II cells

Background

LysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.

Methods

Mouse lung cells were sorted on the basis of CD45^negCD31^negEpCAM^posLysoTracker^pos expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay. To track AT2 cell differentiation, lung epithelial stem and progenitor cells were cultured in a CFU-Epi assay with LysoTracker-supplemented media.

Results

The purity of sorted AT2 cells as determined by SP-C staining was 97.4% and viability was 85.3%. LysoTracker^pos AT2 cells generated SP-C^pos alveolar epithelial cell colonies in culture, and when added to the CFU-Epi culture medium, LysoTracker marked the differentiation of stem/progenitor-derived AT2 cells.

Conclusions

This study describes a novel method for isolating AT2 cells from mouse lungs. The high purity and viability of cells attained by this method, makes them suitable for functional analysis in vitro. The application of LysoTracker to live cell cultures will allow better assessment of the cellular and molecular mechanisms that regulate AT2 cell differentiation.     

An improved method for large scale purification of human holo-transcobalamin II

25 mg of human holo-transcobalamin II with a specific cobalamin-binding capacity of 0.95 mol cobalamin/mol TC II was purified from 122 kg Cohn fraction III with a yield of 73% and a purification factor of 9.34 · 10⁵. Consecutive purification steps comprised CM-Sephadex batchwise ion-exchange chromatography, affinity chromatography, using cyanocobalamin as a ligand, thermolabilly attached to 3.3′-diaminodipropylamine-substituted CH-Sepharose, and gel filtration. The high yield of the purification procedure was achieved by improving the stability of apo-transcobalamin II in the eluate of the CM-Sephadex, and by a few other modifications of a former procedure. In the latter, rapid denaturation of apo-transcobalamin II prohibited the use of long term affinity chromatography, which is obligatory for processing large amounts of Cohn fraction. In addition, subfractionation of transcobalamin II into smaller fragments which occurred in SDS-polyacrylamide gel electrophoresis in previous studies, was now reduced, indicating that proteolysis in the CM-Sephadex eluate had been prevented effectively.     

Identification of calcium-dependent phospholipid-binding proteins (annexins) from guinea pig alveolar type II cells

A new group of calcium-regulating proteins, called annexins or Ca⁺⁺-dependent phospholipid-binding proteins (PLBP), have been detected in different species, organs and cell types. In the present study, we have identified and quantitated PLBP from guinea pig lung, lavage fluid and alveolar type II cells to elucidate the possible role of PLBP in lung surfactant biogenesis and secretion. Lungs were lavaged and type II cells from lavaged lung were isolated by elastase digestion and purified by centrifugal elutriation. For the quantitative identification of PLBP, we performed ELISA assays and Western blot analysis by using an antiserum raised in guinea pigs against a pure rabbit lung 36 kDa PLBP. The lavage fluid, cytosol from lung and type II cells contained 784,167 and 435 ng per mg protein, respectively, of PLBP. The SDS-PAGE electrophoretic pattern and Western blot confirmed that all lung samples have band corresponding to a 36 kDa protein. This indicates that both alveolar type II cells and lavage fluid have higher levels of PLBP than whole lung cytosol.     

Isolation and identification of clara cells from rabbit lung

Summary A procedure has been developed for the isolation of nonciliated bronchiolar epithelial cells (Clara cells) from rabbit lung. Following pulmonary lavage to eliminate macrosphages, cells (5% Clara cells) were released by digestion with 0.1% Protease I in HEPES-buffered balanced salt solution containing 0.5 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid instilled through the trachea. These cells were then separated on the basis of size using the Beckman JE-6 elutriator rotor. The fourth fraction collected from the elutriator contained about 30% Clara cells. This fraction was then layered on a two-polymer aqueous phase system consisting of 5% dextran T500 (DT) and 3.8% polyethylene glycol 6000 (PEG) in sodium phosphate buffer. A cell fraction was obtained from the PEG phase, which included approximately 70% Clara cells. These cells were found to be greater than 90% viable by trypan blue dye exclusion. Identification of isolated Clara cells was confirmed by light microscopic observation of nitro blue tetrazolium staining and by ultrastructural characteristics as observed by electron microscopy. This research was supported in part by the U.S. Environmental Protection Agency under an interagency agreement relating to the Federal Interagency Energy/Environmental Research and Development Program.     

An improved method for the isolation of total RNA from spurce tissues

After many unsuccessful attempts to obtain biologically active mRNAs from spruce (Picea abies) tissues using available protocols, we have adapted a procedure for the isolation of RNAs from needles, shoots, and callus ofPicea species. Our modifications permit the recovery of and an average of 300 μg RNA per g of needles that is suitable for translationin vitro, northern hybridizations, and the construction of cDNA libraries.     

An improved method for the rapid isolation of chromosomal DNA from Mycoplasma spp

A method is described for the rapid isolation of chromosomal deoxyribonucleic acid from species of the genus Mycoplasma. The method involves incubation of washed cells at elevated temperature in the presence of an ionic detergent, chelating agents, and proteinase K prior to the removal of residual protein and ribonucleic acid with ribonuclease and chloroform. It results in a good yield of high molecular weight material that is shown to be free of endogenous nuclease and substantially free of protein or ribonucleic acid contamination without the use of phenol. The isolated DNA is shown to be an excellent substrate for restriction endonuclease digestion and ligation with T4 DNA ligase.     

An improved method for isolation of mutator mutants from mouse FM3A cells and their characterization

An improved method to select mutator mutants was developed. By this new method, mutator mutants were isolated efficiently, and 7 mutants were obtained from cultured mouse FM3A cells. These mutator mutants have an elevated rate of spontaneous mutation at 3 genetic loci (resistance to ouabain, blasticidin S, and tunicamycin). The sensitivity of these mutants to aphidicolin and arabinofuranosylcytosine was the same as in the wild-type cells. Determination of the size of the cellular dNTP pool revealed that there was no large imbalance in the precursor pool in the mutator mutants. These results suggested that the mutator character may be due to alteration in some factor(s) correlated directly to DNA replication. Also, there was no change in the sensitivity of all these mutator mutants to DNA damaging agents.     

一种改进的人体心房肌细胞分离方法

本研究旨在探讨稳定的人体心房肌细胞分离方法,并观察分离的正常窦性心律（normal sinus rhythm,NSR）患者右心房肌细胞基本电生理特性。XXIV型蛋白酶和V型胶原酶两步法进行单个人体心房肌细胞分离,常规全细胞膜片钳技术记录正常的L-型钙通道电流（L-type calcium current,ICa-L）、钠通道电流（sodiumcurrent,INa）、瞬时外向钾通道电流（transient outward potassium current,Itol）和内向整流钾通道电流（inward rectifier potassium current,Ik1）。此方法分离的人体心房肌细胞数量多,细胞膜光滑,折光性强,横纹清楚,耐钙,可用于膜片钳实验的占分离细胞总数的50％-60％。该方法简单、稳定、可靠,酶用量少,分离的心肌细胞质量好,数量多,并能记录到多种离子通道电流,表明其具有正常的电生理功能,适合膜片钳实验。     

Immunofluorescence localization of the regulatory subunit type II of cAMP-dependent protein kinase in PC12 and 3T3 cells in different proliferative states

Localization of the regulatory subunit of cAMP-dependent protein kinase type II was studied in proliferating and quiescent fibroblasts 3T3 and in a cell line of neural origin pheochromocytoma PC12. In actively proliferating PCl2 cells the regulatory subunit was found to be localized in the nucleus. Transition of these cells into a quiescent state was accompanied by a regulatory subunit translocation to the cytoplasm. In 3T3 cells the regulatory subunit was localized in the cytoplasm both in the quiescent and proliferating (though less actively than PC12 cells) states. Similar results were obtained both with monoclonal antibodies and with rabbit monospecific antiserum raised against the regulatory subunit type II from pig brain.     

Vitamin E deficiency sensitizes alveolar type II cells for apoptosis

Pre-term neonates and neonates in general exhibit physiological vitamin E deficiency and are at increased risk for the development of acute lung diseases. Apoptosis is a major cause of acute lung damage in alveolar type II cells. In this paper, we evaluated the hypothesis that vitamin E deficiency predisposes alveolar type II cells to apoptosis. Therefore, we measured markers of apoptosis in alveolar type II cells isolated from control rats, vitamin E deficient rats and deficient rats that were re-fed a vitamin E-enriched diet. Bax and cytosolic cytochrome c increased, and the mitochondrial transmembrane potential and Hsp25 expression was reduced in vitamin E deficiency. Furthermore, increased DNA-fragmentation and numbers of early and late apoptotic cells were seen, but caspases 3 and 8 activities and expression of Fas, Bcl-2, Bcl-x and p53 remained unchanged. Vitamin E depletion did not change the GSH/GSSG ratio and the activities of antioxidant enzymes. Thus, vitamin E deficiency may induce a reversible pro-apoptotic response in lung cells and sensitise them for additional insult. In agreement with this hypothesis, we demonstrate that in vivo hyperoxia alone does not induce apoptosis in type II cells of control rats but reversibly increases DNA-fragmentation and numbers of early apoptotic type II cells in vitamin E-depleted cells.