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Distinguishing Mast Cell Progenitors from Mature Mast Cells in Mice.

Dahlin JS, Ding Z, Hallgren J - Stem Cells Dev. (2015)

Bottom Line: The maturation statuses of the cells were confirmed using three main strategies: (1) MCp, but not mature mast cells, were shown to be depleted by sublethal whole-body γ-irradiation. (2) The MCp were small and immature in terms of granule formation, whereas the mature mast cells were larger and had fully developed metachromatic granules. (3) The MCp had fewer transcripts of mast cell-specific proteases and the enzyme responsible for sulfation of heparin than mature mast cells.Moreover, isolated peritoneal MCp gave rise to mast cells when cultured in vitro.Using this strategy, mast cell maturation can be studied in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University , Uppsala, Sweden .

ABSTRACT
Mast cells originate from the bone marrow and develop into c-kit(+) FcɛRI(+) cells. Both mast cell progenitors (MCp) and mature mast cells express these cell surface markers, and ways validated to distinguish between the two maturation forms with flow cytometry have been lacking. Here, we show that primary peritoneal MCp from naïve mice expressed high levels of integrin β7 and had a low side scatter (SSC) light profile; whereas mature mast cells expressed lower levels of integrin β7 and had a high SSC light profile. The maturation statuses of the cells were confirmed using three main strategies: (1) MCp, but not mature mast cells, were shown to be depleted by sublethal whole-body γ-irradiation. (2) The MCp were small and immature in terms of granule formation, whereas the mature mast cells were larger and had fully developed metachromatic granules. (3) The MCp had fewer transcripts of mast cell-specific proteases and the enzyme responsible for sulfation of heparin than mature mast cells. Moreover, isolated peritoneal MCp gave rise to mast cells when cultured in vitro. To summarize, we have defined MCp and mature mast cells in naïve mice by flow cytometry. Using this strategy, mast cell maturation can be studied in vivo.

No MeSH data available.


Related in: MedlinePlus

Cells from naïve mouse lungs were analyzed by flow cytometry. CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi cells expressed FcɛRI and had a low SSC light profile, suggesting that these cells were MCp. CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7−/lo/int cells also expressed FcɛRI, but had a high SSC light profile, indicating that that they were mature mast cells. Pooled cells from three to four mice were analyzed. The graphs are derived from one out of two similar experiments. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the number of CD45+ lung cells.
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f5: Cells from naïve mouse lungs were analyzed by flow cytometry. CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi cells expressed FcɛRI and had a low SSC light profile, suggesting that these cells were MCp. CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7−/lo/int cells also expressed FcɛRI, but had a high SSC light profile, indicating that that they were mature mast cells. Pooled cells from three to four mice were analyzed. The graphs are derived from one out of two similar experiments. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the number of CD45+ lung cells.

Mentions: A similar gating strategy to the one used to distinguish peritoneal MCp from mature mast cells was applied to lung cells. In addition to the markers used in peritoneum, CD45 was used to exclude stromal cells from the analysis. In naïve mice, nonapoptotic CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi FcɛRI+ SSClo MCp constituted only 0.0040% of the CD45+ lung cells (Fig. 5). Mature mast cells, identified as CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7−/lo/int FcɛRI+ SSChi cells, were also rare and constituted 0.0050% of the CD45+ lung cells (Fig. 5).


Distinguishing Mast Cell Progenitors from Mature Mast Cells in Mice.

Dahlin JS, Ding Z, Hallgren J - Stem Cells Dev. (2015)

Cells from naïve mouse lungs were analyzed by flow cytometry. CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi cells expressed FcɛRI and had a low SSC light profile, suggesting that these cells were MCp. CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7−/lo/int cells also expressed FcɛRI, but had a high SSC light profile, indicating that that they were mature mast cells. Pooled cells from three to four mice were analyzed. The graphs are derived from one out of two similar experiments. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the number of CD45+ lung cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4499794&req=5

f5: Cells from naïve mouse lungs were analyzed by flow cytometry. CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi cells expressed FcɛRI and had a low SSC light profile, suggesting that these cells were MCp. CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7−/lo/int cells also expressed FcɛRI, but had a high SSC light profile, indicating that that they were mature mast cells. Pooled cells from three to four mice were analyzed. The graphs are derived from one out of two similar experiments. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the number of CD45+ lung cells.
Mentions: A similar gating strategy to the one used to distinguish peritoneal MCp from mature mast cells was applied to lung cells. In addition to the markers used in peritoneum, CD45 was used to exclude stromal cells from the analysis. In naïve mice, nonapoptotic CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi FcɛRI+ SSClo MCp constituted only 0.0040% of the CD45+ lung cells (Fig. 5). Mature mast cells, identified as CD45+ Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7−/lo/int FcɛRI+ SSChi cells, were also rare and constituted 0.0050% of the CD45+ lung cells (Fig. 5).

Bottom Line: The maturation statuses of the cells were confirmed using three main strategies: (1) MCp, but not mature mast cells, were shown to be depleted by sublethal whole-body γ-irradiation. (2) The MCp were small and immature in terms of granule formation, whereas the mature mast cells were larger and had fully developed metachromatic granules. (3) The MCp had fewer transcripts of mast cell-specific proteases and the enzyme responsible for sulfation of heparin than mature mast cells.Moreover, isolated peritoneal MCp gave rise to mast cells when cultured in vitro.Using this strategy, mast cell maturation can be studied in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University , Uppsala, Sweden .

ABSTRACT
Mast cells originate from the bone marrow and develop into c-kit(+) FcɛRI(+) cells. Both mast cell progenitors (MCp) and mature mast cells express these cell surface markers, and ways validated to distinguish between the two maturation forms with flow cytometry have been lacking. Here, we show that primary peritoneal MCp from naïve mice expressed high levels of integrin β7 and had a low side scatter (SSC) light profile; whereas mature mast cells expressed lower levels of integrin β7 and had a high SSC light profile. The maturation statuses of the cells were confirmed using three main strategies: (1) MCp, but not mature mast cells, were shown to be depleted by sublethal whole-body γ-irradiation. (2) The MCp were small and immature in terms of granule formation, whereas the mature mast cells were larger and had fully developed metachromatic granules. (3) The MCp had fewer transcripts of mast cell-specific proteases and the enzyme responsible for sulfation of heparin than mature mast cells. Moreover, isolated peritoneal MCp gave rise to mast cells when cultured in vitro. To summarize, we have defined MCp and mature mast cells in naïve mice by flow cytometry. Using this strategy, mast cell maturation can be studied in vivo.

No MeSH data available.


Related in: MedlinePlus