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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. 1Flow cytometry SSC and FFC dot plots of seabream intestine cells showing three subpopulations (R1, R2 and R3). (a) Representative dot plot of a S2 suspension obtained after isolation of cells using both chemical (DTT, 10 min) and enzymatic (collagenase 0.15 mg/ml, 60 min) treatments. (b) Representative dot plot obtained after the same suspension in Figure 1a is passed through a nylon wool column (NW) for 60 min. (c) Representative dot plot of a total suspension obtained by mechanical stripping,
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f1: Fig. 1Flow cytometry SSC and FFC dot plots of seabream intestine cells showing three subpopulations (R1, R2 and R3). (a) Representative dot plot of a S2 suspension obtained after isolation of cells using both chemical (DTT, 10 min) and enzymatic (collagenase 0.15 mg/ml, 60 min) treatments. (b) Representative dot plot obtained after the same suspension in Figure 1a is passed through a nylon wool column (NW) for 60 min. (c) Representative dot plot of a total suspension obtained by mechanical stripping,

Mentions: Identification of GALT cell subsets according to the FSC and SSC values obtained by flow cytometry was not easy because the dot plots showed no well defined subsets. Three cell subsets of variable relative abundance could be distinguished, such variations appearing not to be associated to a particular purification protocol but to differences between specimens. In general, the majority of cells corresponded to low FSC and low SSC values (R1). A second subpopulation, R2, consisted of medium FSC values but with relatively high spread and medium SSC values. Finally, a small population (R3) of low FSC and low to high SSC values was found (Fig. 1A).


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. 1Flow cytometry SSC and FFC dot plots of seabream intestine cells showing three subpopulations (R1, R2 and R3). (a) Representative dot plot of a S2 suspension obtained after isolation of cells using both chemical (DTT, 10 min) and enzymatic (collagenase 0.15 mg/ml, 60 min) treatments. (b) Representative dot plot obtained after the same suspension in Figure 1a is passed through a nylon wool column (NW) for 60 min. (c) Representative dot plot of a total suspension obtained by mechanical stripping,
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211574&req=5

f1: Fig. 1Flow cytometry SSC and FFC dot plots of seabream intestine cells showing three subpopulations (R1, R2 and R3). (a) Representative dot plot of a S2 suspension obtained after isolation of cells using both chemical (DTT, 10 min) and enzymatic (collagenase 0.15 mg/ml, 60 min) treatments. (b) Representative dot plot obtained after the same suspension in Figure 1a is passed through a nylon wool column (NW) for 60 min. (c) Representative dot plot of a total suspension obtained by mechanical stripping,
Mentions: Identification of GALT cell subsets according to the FSC and SSC values obtained by flow cytometry was not easy because the dot plots showed no well defined subsets. Three cell subsets of variable relative abundance could be distinguished, such variations appearing not to be associated to a particular purification protocol but to differences between specimens. In general, the majority of cells corresponded to low FSC and low SSC values (R1). A second subpopulation, R2, consisted of medium FSC values but with relatively high spread and medium SSC values. Finally, a small population (R3) of low FSC and low to high SSC values was found (Fig. 1A).

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