Use of commercially available cell culture inserts for primary culture and electrophysiologic studies of guinea pig gastric mucous epithelial cells

Rutten, Michael J.

doi:10.1007/bf01409016

Cited by 3 publications

(1 citation statement)

References 62 publications

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“…After fura 2 loading, the gastric cultures were transferred to a horizontal open perfusion chamber that had been modified to hold either a permeable Falcon filter or a glass coverslip (46). The chamber was then placed on the stage of a Nikon Diaphot TMD inverted microscope equipped with a fluorescence objective (Nikon Fluor-phase-3DM, numerical aperature 60/0.7, 160-mm working distance).…”

Section: Methodsmentioning

confidence: 99%

Effects ofHelicobacter pylorion intracellular Ca²⁺signaling in normal human gastric mucous epithelial cells

Marlink

Bacon

Sheppard

et al. 2003

American Journal of Physiology-Gastrointestinal and Liver Physi

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In stomach, Helicobacter pylori (Hp) adheres to gastric mucous epithelial cells (GMEC) and initiates several different signal transduction events. Alteration of intracellular Ca2+ concentration ([Ca2+]i) is an important signaling mechanism in numerous bacteria-host model systems. Changes in [Ca2+]i induced by Hp in normal human GMEC have not yet been described; therefore, we examined effects of Hp on [Ca2+]i in normal human GMEC and a nontransformed GMEC line (HFE-145). Cultured cells were grown on glass slides, porous filters, or 96-well plates and loaded with fura 2 or fluo 4. Hp wild-type strain 60190 and vacA-, cagA-, and picB-/cagE- isogenic mutants were incubated with cells. Changes in [Ca2+]i were recorded with a fluorimeter or fluorescence plate reader. Wild-type Hp produced dose-dependent biphasic transient [Ca2+]i peak and plateau changes in both cell lines. Hp vacA- isogenic mutant produced changes in [Ca2+]i similar to those produced by wild type. Compared with wild type, cagA- and picB-/cagE- isogenic mutants produced lower peak changes and did not generate a plateau change. Preloading cultures with intracellular Ca2+ chelator BAPTA blocked all Hp-induced [Ca2+]i changes. Thapsigargin pretreatment of cultures to release Ca2+ from internal stores reduced peak change. Extracellular Ca2+ removal reduced plateau response. Hp-induced peak response was sensitive to G proteins and PLC inhibitors. Hp-induced plateau change was sensitive to G protein inhibitors, src kinases, and PLA2. These findings are the first to show that H. pylori alters [Ca2+]i in normal GMEC through a Ca2+ release/influx mechanism that depends on expression of cagA and picB/cagE genes.

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Section: Methodsmentioning

confidence: 99%

Effects ofHelicobacter pylorion intracellular Ca²⁺signaling in normal human gastric mucous epithelial cells

Marlink

Bacon

Sheppard

et al. 2003

American Journal of Physiology-Gastrointestinal and Liver Physi

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Kelly

Fletcher

Pärt

et al. 2000

Methods in Cell Science

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Techniques for the in vitro 'reconstruction' of freshwater rainbow trout branchial epithelia using the primary culture of gill cells on permeable polyethylene terephthalate cell culture filter supports are described. Representing models of the freshwater fish gill, epithelia grown by two separate techniques are composed of branchial pavement cells with or without the inclusion of mitochondria-rich (MR) cells. The generation of epithelia consisting of pavement cells only (via a method called single seeded inserts = SSI) involves an initial period of flask culture during which time MR cells, that appear unable to attach to the culture flask base, are excluded from the general cell populace. Alternately, the generation of a heterogeneous epithelia consisting of both pavement cells and MR cells (via a method called double seeded inserts = DSI) is facilitated by the direct seeding of cells into cell culture filter inserts. Critical to this second procedure is the repeat seeding of filter inserts over a two day period. Repeat seeding appears to allow MR cells to nest amongst the attached cell layer generated by the first day's seeding. The use of cell culture filter supports allows free access to both the apical and basolateral compartment of the epithelium and is ideal for experimental manipulation. Cells are grown under symmetrical conditions (apical media/basolateral media) and epithelium growth is measured as a function of transepithelial resistance (TER). When the epithelia exhibit a plateau in growth they can be subjected to asymmetrical conditions (freshwater apical/media basolateral) in order to assess gill cell function as in vivo.

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Cultured Branchial Epithelia From Freshwater Fish Gills

Wood

Pärt

1997

Journal of Experimental Biology

114

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We have developed a method for the primary culture of gill epithelial cells from freshwater rainbow trout on permeable supports, polyethylene terephthalate membranes ('filter inserts'). Primary cultures of gill cells (6-9 days in Leibowitz L-15 culture medium plus foetal bovine serum and glutamine) are trypsinized and the cells seeded onto the inserts. After 6 days of growth with L-15 medium on both surfaces (approximately isotonic to trout plasma), the cells form a tight epithelium as judged from a progressive rise in transepithelial resistance which reaches a stable plateau for a further 6 days, as long as L-15 exposure is continued on both surfaces. The cultured epithelium (approximately 8 µm thick) typically consists of 2-4 overlapping cell layers organized as in the lamellae in vivo, with large intercellular spaces, multiple desmosomes and putative tight junctions. The cells appear to be exclusively pavement-type cells with an apical surface glycocalyx, an abundance of rough endoplasmic reticulum, no selective DASPEI staining and relatively few mitochondria. Transepithelial resistance (approximately 3.5 k cm2), permeability to a paracellular marker (polyethylene glycol-4000; 0.17x10(-6) cm s-1) and unidirectional flux of Na+ and Cl- (approximately 300 nmol cm-2 h-1) all appear realistic because they compare well with in vivo values; net fluxes of Na+ and Cl- are zero. The preparation acidifies the apical medium, which accumulates a greater concentration of ammonia. Upon exposure to apical freshwater, resistance increases six- to elevenfold and a basolateral-negative transepithelial potential (TEP) develops as in vivo. These responses occur even when mannitol is used to prevent changes in apical osmotic pressure. Net Na+ and Cl- loss rates are low over the first 12 h (-125 nmol cm-2 h-1) but increase substantially by 48 h. The elevated resistance and negative TEP gradually attenuate but remain significantly higher than pre-exposure values after 48 h of apical freshwater exposure. The preparation may provide a valuable new tool for characterizing some of the mechanisms of active and passive ion transport in the pavement cells of the freshwater gill.

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Use of commercially available cell culture inserts for primary culture and electrophysiologic studies of guinea pig gastric mucous epithelial cells

Cited by 3 publications

References 62 publications

Effects ofHelicobacter pylorion intracellular Ca²⁺signaling in normal human gastric mucous epithelial cells

Effects ofHelicobacter pylorion intracellular Ca²⁺signaling in normal human gastric mucous epithelial cells

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Cultured Branchial Epithelia From Freshwater Fish Gills

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Use of commercially available cell culture inserts for primary culture and electrophysiologic studies of guinea pig gastric mucous epithelial cells

Cited by 3 publications

References 62 publications

Effects ofHelicobacter pylorion intracellular Ca2+signaling in normal human gastric mucous epithelial cells

Effects ofHelicobacter pylorion intracellular Ca2+signaling in normal human gastric mucous epithelial cells

Untitled

Cultured Branchial Epithelia From Freshwater Fish Gills

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Effects ofHelicobacter pylorion intracellular Ca²⁺signaling in normal human gastric mucous epithelial cells

Effects ofHelicobacter pylorion intracellular Ca²⁺signaling in normal human gastric mucous epithelial cells