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Assessment of different protocols for the isolation and purification of gut associated lymphoid cells from the gilthead seabream (Sparus aurata L.).

Salinas I, Meseguer J, Esteban MA - Biol Proced Online (2007)

Bottom Line: Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations.Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some lymphocytes.Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells, reflecting results from light microscopy and reports in other teleost species.

View Article: PubMed Central - PubMed

Affiliation: Fish Innate Immune System Group, Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain.

ABSTRACT
Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some lymphocytes. Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells, reflecting results from light microscopy and reports in other teleost species. The present data set up the basis for future functional characterization of GALT in seabream.

No MeSH data available.


Related in: MedlinePlus

Fig. 4Light micrographs of semithin sections of seabream intestine at different stages of cell extraction. L (lumen), E (enterocytes), LP (lamina propria). (a) Gut semithin section at the beginning of the isolation procedure (control sample, time 0). Bar: 10 μm. (b) Gut semithin section after 10min in DTT. Bar: 10 μm (c) Gut semithin section after 10 min in DTT and 60 min in collagenase (0.15 mg/ml). Note the open interepithelial spaces (arrows) that allow leucocytes to be freed into the media. Bar: 10 μm.
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f4: Fig. 4Light micrographs of semithin sections of seabream intestine at different stages of cell extraction. L (lumen), E (enterocytes), LP (lamina propria). (a) Gut semithin section at the beginning of the isolation procedure (control sample, time 0). Bar: 10 μm. (b) Gut semithin section after 10min in DTT. Bar: 10 μm (c) Gut semithin section after 10 min in DTT and 60 min in collagenase (0.15 mg/ml). Note the open interepithelial spaces (arrows) that allow leucocytes to be freed into the media. Bar: 10 μm.

Mentions: Observation of semithin sections of the seabream gut tissue during the different steps of the isolation protocol permitted visualisation of the different degrees of digestion of the tissue as well as the progressive release of immune cells. Treatment with DTT loosened the spaces between mucosal epithelium enterocytes (Fig. 4B) compared to control fragments (Fig. 4A), whereas treatment with collagenase for 60 minutes sufficed to digest the connective tissue that forms the lamina propria of the intestinal epithelium (Fig. 4C).


Assessment of different protocols for the isolation and purification of gut associated lymphoid cells from the gilthead seabream (Sparus aurata L.).

Salinas I, Meseguer J, Esteban MA - Biol Proced Online (2007)

Fig. 4Light micrographs of semithin sections of seabream intestine at different stages of cell extraction. L (lumen), E (enterocytes), LP (lamina propria). (a) Gut semithin section at the beginning of the isolation procedure (control sample, time 0). Bar: 10 μm. (b) Gut semithin section after 10min in DTT. Bar: 10 μm (c) Gut semithin section after 10 min in DTT and 60 min in collagenase (0.15 mg/ml). Note the open interepithelial spaces (arrows) that allow leucocytes to be freed into the media. Bar: 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211574&req=5

f4: Fig. 4Light micrographs of semithin sections of seabream intestine at different stages of cell extraction. L (lumen), E (enterocytes), LP (lamina propria). (a) Gut semithin section at the beginning of the isolation procedure (control sample, time 0). Bar: 10 μm. (b) Gut semithin section after 10min in DTT. Bar: 10 μm (c) Gut semithin section after 10 min in DTT and 60 min in collagenase (0.15 mg/ml). Note the open interepithelial spaces (arrows) that allow leucocytes to be freed into the media. Bar: 10 μm.
Mentions: Observation of semithin sections of the seabream gut tissue during the different steps of the isolation protocol permitted visualisation of the different degrees of digestion of the tissue as well as the progressive release of immune cells. Treatment with DTT loosened the spaces between mucosal epithelium enterocytes (Fig. 4B) compared to control fragments (Fig. 4A), whereas treatment with collagenase for 60 minutes sufficed to digest the connective tissue that forms the lamina propria of the intestinal epithelium (Fig. 4C).

Bottom Line: Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations.Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some lymphocytes.Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells, reflecting results from light microscopy and reports in other teleost species.

View Article: PubMed Central - PubMed

Affiliation: Fish Innate Immune System Group, Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain.

ABSTRACT
Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some lymphocytes. Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells, reflecting results from light microscopy and reports in other teleost species. The present data set up the basis for future functional characterization of GALT in seabream.

No MeSH data available.


Related in: MedlinePlus