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Effects of IL-3 and SCF on Histamine Production Kinetics and Cell Phenotype in Rat Bone Marrow-derived Mast Cells.

Lee HN, Kim CH, Song GG, Cho SW - Immune Netw (2010)

Bottom Line: However, both RMCP II production and histamine synthesis peaked after 11 days of culture.By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5.By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Korea University Graduate School, Seoul 136-705, Korea.

ABSTRACT

Background: Rat mast cells were regarded as a good model for mast cell function in immune response.

Methods: Rat bone marrow mast cells (BMMC) were prepared both by recombinant rat IL-3 (rrIL-3) and by recombinant mouse stem cell factor (rmSCF), and investigated for both proliferation and differentiation in time course. Rat BMMC was induced by culture of rat bone marrow cells (BMCs) in the presence of both rrIL-3 (5 ng/ml) and rmSCF (5 ng/ml). Culture media were changed 2 times per week with the cell number condition of 5x10(4)/ml in 6 well plate. Proliferation was analyzed by cell number and cell counting kit-8 (CCK-8) and differentiation was by rat mast cell protease (RMCP) II and histamine.

Results: Cell proliferation rates reached a maximum at 8 or 11 days of culture and decreased thereafter. However, both RMCP II production and histamine synthesis peaked after 11 days of culture. By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5. By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules. Using gated flow cytometry, we showed that cultured BMCs expressed high levels of FcepsilonRI and the mast cell antigen, ganglioside, on culture day 11.

Conclusion: These results indicate that rat BMMCs were generated by culturing BMCs in the presence of rrIL-3 and rmSCF and that the BMMCs have the characteristics of mucosal mast cells.

No MeSH data available.


Related in: MedlinePlus

TEM of rat bone marrow cells. Rat bone marrow cells cultured in the presence of rrIL-3 and rmSCF were analyzed by TEM at day 0 (A), day 4 (B), day 11 (C), and day 15 (D).
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Figure 2: TEM of rat bone marrow cells. Rat bone marrow cells cultured in the presence of rrIL-3 and rmSCF were analyzed by TEM at day 0 (A), day 4 (B), day 11 (C), and day 15 (D).

Mentions: To understand the detailed structure of cultured cells, cells were assayed by TEM. Rat bone marrow cells that were harvested over a 70% Percoll cushion on day 0 were very diverse in size and intracellular structure. The outer cell membrane was relatively irregular (Fig. 2A). At day 4, round cells with large nuclei were relatively predominant. Cells with dense cytoplasm, as seen by round black spots, were seen intermittently. At day 11, the size of the nuclei was relatively smaller than that of day 4 cells. The amount of cytoplasm was greater at day 11 than that at day 4. At day 15, the cells with dense cytoplasm were rare, and open or empty cytoplasmic structures were observed.


Effects of IL-3 and SCF on Histamine Production Kinetics and Cell Phenotype in Rat Bone Marrow-derived Mast Cells.

Lee HN, Kim CH, Song GG, Cho SW - Immune Netw (2010)

TEM of rat bone marrow cells. Rat bone marrow cells cultured in the presence of rrIL-3 and rmSCF were analyzed by TEM at day 0 (A), day 4 (B), day 11 (C), and day 15 (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2837153&req=5

Figure 2: TEM of rat bone marrow cells. Rat bone marrow cells cultured in the presence of rrIL-3 and rmSCF were analyzed by TEM at day 0 (A), day 4 (B), day 11 (C), and day 15 (D).
Mentions: To understand the detailed structure of cultured cells, cells were assayed by TEM. Rat bone marrow cells that were harvested over a 70% Percoll cushion on day 0 were very diverse in size and intracellular structure. The outer cell membrane was relatively irregular (Fig. 2A). At day 4, round cells with large nuclei were relatively predominant. Cells with dense cytoplasm, as seen by round black spots, were seen intermittently. At day 11, the size of the nuclei was relatively smaller than that of day 4 cells. The amount of cytoplasm was greater at day 11 than that at day 4. At day 15, the cells with dense cytoplasm were rare, and open or empty cytoplasmic structures were observed.

Bottom Line: However, both RMCP II production and histamine synthesis peaked after 11 days of culture.By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5.By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Korea University Graduate School, Seoul 136-705, Korea.

ABSTRACT

Background: Rat mast cells were regarded as a good model for mast cell function in immune response.

Methods: Rat bone marrow mast cells (BMMC) were prepared both by recombinant rat IL-3 (rrIL-3) and by recombinant mouse stem cell factor (rmSCF), and investigated for both proliferation and differentiation in time course. Rat BMMC was induced by culture of rat bone marrow cells (BMCs) in the presence of both rrIL-3 (5 ng/ml) and rmSCF (5 ng/ml). Culture media were changed 2 times per week with the cell number condition of 5x10(4)/ml in 6 well plate. Proliferation was analyzed by cell number and cell counting kit-8 (CCK-8) and differentiation was by rat mast cell protease (RMCP) II and histamine.

Results: Cell proliferation rates reached a maximum at 8 or 11 days of culture and decreased thereafter. However, both RMCP II production and histamine synthesis peaked after 11 days of culture. By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5. By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules. Using gated flow cytometry, we showed that cultured BMCs expressed high levels of FcepsilonRI and the mast cell antigen, ganglioside, on culture day 11.

Conclusion: These results indicate that rat BMMCs were generated by culturing BMCs in the presence of rrIL-3 and rmSCF and that the BMMCs have the characteristics of mucosal mast cells.

No MeSH data available.


Related in: MedlinePlus