Isolation and culture of alveolar type II cells

Dobbs, Leland G.

doi:10.1152/ajplung.1990.258.4.l134

Cited by 285 publications

(300 citation statements)

References 84 publications

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“…Isolation and culture of rat AECII were performed using the methods reported previously. 34 AECII were isolated from Sprague-Dawley female rats by 'panning'. 34 Yields were around 15 Â 10 6 AECII cells/rat and purity was around 90-95% after 1 day culture in plastics.…”

Section: Cell Culturementioning

confidence: 99%

“…34 AECII were isolated from Sprague-Dawley female rats by 'panning'. 34 Yields were around 15 Â 10 6 AECII cells/rat and purity was around 90-95% after 1 day culture in plastics. Phenotypes of AECII were identified by three methods: (a) alkaline phosphatase staining, 35 (b) tannic acid staining, and (c) surfactant protein (SP-C) determined by immunochemistry staining.…”

Section: Cell Culturementioning

confidence: 99%

“…RT-PCR was performed using the Titan one-step RT-PCR kit (Roche Molecular Biochemicals) with samples of 0.2 mg of total RNA as templates. The thermocycling parameters were composed of an initial cycle at 501C for 30 min for synthesizing the first strand DNA, and then 951C for 180 s followed by 30 cycles at 951C for 60 s, 551C for 60 s and 721C for 60 s. Several lengths of cycles 26,30,34 have been tested, and 30 cycles were used for data presentations. The mRNA level of GAPDH was used as internal control.…”

Section: Rna Extraction and Analysismentioning

confidence: 99%

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Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity

et al. 2005

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It is unknown whether base excision DNA repair (BER) proteins interact with mitogen-activated protein kinases (MAPK) under oxidation. Here, we explored roles of BER proteins in signaling transduction involving MAPK during hyperoxia. We demonstrated that ERK1/2 phosphorylation in A549 cells was increased in 95% O 2 . p38 activity in A549 cells was also increased by exposure to 95% O 2 . To evaluate regulatory roles of MAPK, we have transduced A549 cells and primary alveolar epithelial type II cells (AECII) to overexpress 8-oxoguanine DNA glycosylase (hOgg1). Overexpression of hOgg1 reduced hyperoxic toxicity in A549 and AECII cells. Furthermore, protection by BER against hyperoxia appeared to involve an upregulation of ERK1/2 and downregulation of p38. These observations demonstrate, for the first time, that reduction of hyperoxic toxicity by BER proteins may be involved with MAPK activity, thereby impacting cell survival. Furthermore, our studies suggest that modulation of MAPK may be used in combination with BER proteins to counteract hyperoxic toxicity.

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Section: Cell Culturementioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Section: Rna Extraction and Analysismentioning

confidence: 99%

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Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity

et al. 2005

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“…Alveolar type 2 cells were isolated as described previously [44,45]. In brief, untreated animals were killed by exsanguination via the descending aorta in a deep intraperitoneal anesthesia as described above.…”

Section: Preparation Of At-2 Epithelial Cellsmentioning

confidence: 99%

A Role for Forkhead Box A1 in Acute Lung Injury

et al. 2009

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Abstract-Forkhead box protein A1 (FoxA1) is an evolutionarily conserved winged helix transcription factor with diverse regulatory functions. However, little is known about the role of FoxA1 in acute lung injury (ALI) and pulmonary cell injury. In this study, an in vivo model was employed whereby rats were administered an intravenous injection of oleic acid (OA, 0.1 ml/kg), and alveolar type II epithelial cells (AT-2 cells) injury was induced by hydrogen peroxide (H 2 O 2 ) in vitro. OA injection resulted in lung injury and AT-2 cells apoptosis in vivo. OA injection and H 2 O 2 upregulated FoxA1 mRNA and protein in lung tissue of the in vivo ALI model and in H 2 O 2 challenged AT-2 cells. Overexpression of FoxA1 promoted apoptosis, whereas FoxA1 deficiency, induced by antisense oligonucleotides, decreased AT-2 cells apoptosis induced by H 2 O 2 , as shown by flow cytometry. These results suggest that FoxA1 may play an important role in ALI by promoting apoptosis of pulmonary epithelial cells.KEY WORDS: acute lung injury; forkhead box A1; apoptosis.

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“…Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14,15 , flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs.…”

Section: Introductionmentioning

confidence: 99%

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Gereke

Autengruber

Gröbe

et al. 2012

JoVE

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Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigenpresenting cells in both infection and T-cell mediated autoimmunity [1][2][3][4][5][6][7][8] . Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines [9][10][11][12] . Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13 . Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSC high ) cell population and are separated by fluorescence activated cell sorting 3 .In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14,15 , flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome ...

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Isolation and culture of alveolar type II cells

Cited by 285 publications

References 84 publications

Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity

Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity

A Role for Forkhead Box A1 in Acute Lung Injury

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

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