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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

(A, B) Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp and Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells (MC) were doubly sorted by FACS and stained with May-Grünwald Giemsa. Original magnification, 40×. The width of each photo corresponds to 32 μm. (C, D) The diameter and the metachromatic area of the MCp and the mature mast cells were measured. Each dot in (C, D) represents one cell. The means and SEM are shown. The cells in (A–D) were sorted from three to six mice in two separate experiments. Cells from both experiments are shown in the figure. FACS, fluorescence-activated cell sorting. Color images available online at www.liebertpub.com/scd
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f2: (A, B) Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp and Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells (MC) were doubly sorted by FACS and stained with May-Grünwald Giemsa. Original magnification, 40×. The width of each photo corresponds to 32 μm. (C, D) The diameter and the metachromatic area of the MCp and the mature mast cells were measured. Each dot in (C, D) represents one cell. The means and SEM are shown. The cells in (A–D) were sorted from three to six mice in two separate experiments. Cells from both experiments are shown in the figure. FACS, fluorescence-activated cell sorting. Color images available online at www.liebertpub.com/scd

Mentions: To elucidate the appearance of the peritoneal mast cell population, the MCp and the mature mast cells were isolated using FACS and stained with May-Grünwald Giemsa (Fig. 2A, B). The Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp were small, 15.7±0.4 μm (mean±SEM) in diameter, with a round nucleus that filled up a large portion of the cytoplasm (Fig. 2A, C). The Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells were larger, with a diameter of 22.7±0.3 μm (Fig. 2B, C). The MCp contained metachromatic granules (Fig. 2A). However, the mature mast cells had higher numbers of granules, which appeared more densely stained than those of MCp (compare Fig. 2A and B). The difference in metachromatic granule staining between MCp and mature mast cells was quantified using an image analysis software. The mature mast cells had a larger metachromatic area per cell than MCp (Fig. 2D).


Distinguishing Mast Cell Progenitors from Mature Mast Cells in Mice.

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

(A, B) Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp and Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells (MC) were doubly sorted by FACS and stained with May-Grünwald Giemsa. Original magnification, 40×. The width of each photo corresponds to 32 μm. (C, D) The diameter and the metachromatic area of the MCp and the mature mast cells were measured. Each dot in (C, D) represents one cell. The means and SEM are shown. The cells in (A–D) were sorted from three to six mice in two separate experiments. Cells from both experiments are shown in the figure. FACS, fluorescence-activated cell sorting. Color images available online at www.liebertpub.com/scd
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4499794&req=5

f2: (A, B) Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp and Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells (MC) were doubly sorted by FACS and stained with May-Grünwald Giemsa. Original magnification, 40×. The width of each photo corresponds to 32 μm. (C, D) The diameter and the metachromatic area of the MCp and the mature mast cells were measured. Each dot in (C, D) represents one cell. The means and SEM are shown. The cells in (A–D) were sorted from three to six mice in two separate experiments. Cells from both experiments are shown in the figure. FACS, fluorescence-activated cell sorting. Color images available online at www.liebertpub.com/scd
Mentions: To elucidate the appearance of the peritoneal mast cell population, the MCp and the mature mast cells were isolated using FACS and stained with May-Grünwald Giemsa (Fig. 2A, B). The Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp were small, 15.7±0.4 μm (mean±SEM) in diameter, with a round nucleus that filled up a large portion of the cytoplasm (Fig. 2A, C). The Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells were larger, with a diameter of 22.7±0.3 μm (Fig. 2B, C). The MCp contained metachromatic granules (Fig. 2A). However, the mature mast cells had higher numbers of granules, which appeared more densely stained than those of MCp (compare Fig. 2A and B). The difference in metachromatic granule staining between MCp and mature mast cells was quantified using an image analysis software. The mature mast cells had a larger metachromatic area per cell than MCp (Fig. 2D).

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