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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. 3Purification of seabream GALT cells by Percoll density gradients. (a) Representative flow cytometry dot plot of intermediate density band (ID) corresponding to 1.060 g/l. (b) Representative flow cytometry dot plot of cells found at the highest density band (HD) (1.075 g/l). (c) Light micrograph of a Giemsa stained cytocentrifugation of ID band cells. (d) Light micrograph of a Giemsa stained cytocentrifugation of HD band cells.
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f3: Fig. 3Purification of seabream GALT cells by Percoll density gradients. (a) Representative flow cytometry dot plot of intermediate density band (ID) corresponding to 1.060 g/l. (b) Representative flow cytometry dot plot of cells found at the highest density band (HD) (1.075 g/l). (c) Light micrograph of a Giemsa stained cytocentrifugation of ID band cells. (d) Light micrograph of a Giemsa stained cytocentrifugation of HD band cells.

Mentions: Flow cytometry analysis of these bands revealed that the viability of ID and HD was higher than in the original cell suspension since dead cells stayed mainly in LD. Moreover, LD contained a cell subpopulation resembling those found in the S2 and NW pools. The ID band was enriched in cells of intermediate size and low-medium complexity (Fig. 3A). Finally, HD was enriched in a cell populations characterised by both very low FSC and SSC (Fig. 3B).


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. 3Purification of seabream GALT cells by Percoll density gradients. (a) Representative flow cytometry dot plot of intermediate density band (ID) corresponding to 1.060 g/l. (b) Representative flow cytometry dot plot of cells found at the highest density band (HD) (1.075 g/l). (c) Light micrograph of a Giemsa stained cytocentrifugation of ID band cells. (d) Light micrograph of a Giemsa stained cytocentrifugation of HD band cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211574&req=5

f3: Fig. 3Purification of seabream GALT cells by Percoll density gradients. (a) Representative flow cytometry dot plot of intermediate density band (ID) corresponding to 1.060 g/l. (b) Representative flow cytometry dot plot of cells found at the highest density band (HD) (1.075 g/l). (c) Light micrograph of a Giemsa stained cytocentrifugation of ID band cells. (d) Light micrograph of a Giemsa stained cytocentrifugation of HD band cells.
Mentions: Flow cytometry analysis of these bands revealed that the viability of ID and HD was higher than in the original cell suspension since dead cells stayed mainly in LD. Moreover, LD contained a cell subpopulation resembling those found in the S2 and NW pools. The ID band was enriched in cells of intermediate size and low-medium complexity (Fig. 3A). Finally, HD was enriched in a cell populations characterised by both very low FSC and SSC (Fig. 3B).

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