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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

Doubly sorted Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp were cultured with IL-3 and stem cell factor. The cultured cells were analyzed after (A) 5 days, (B) 14 days, and (C, D) 15 days. Flow cytometric analysis was performed in (A, C, D), whereas May-Grünwald Giemsa staining was performed in (B). Primary MCp gated without the use of the SSC light profile (Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi FcɛRI+) were used as day 0 control in (D, black line). The cultured MCp were derived from six naïve mice in (A–D). The day 0 peritoneal MCp gated in (D) were from four naïve mice. The width of the photo in (C) corresponds to 64 μm. Color images available online at www.liebertpub.com/scd
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f3: Doubly sorted Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp were cultured with IL-3 and stem cell factor. The cultured cells were analyzed after (A) 5 days, (B) 14 days, and (C, D) 15 days. Flow cytometric analysis was performed in (A, C, D), whereas May-Grünwald Giemsa staining was performed in (B). Primary MCp gated without the use of the SSC light profile (Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi FcɛRI+) were used as day 0 control in (D, black line). The cultured MCp were derived from six naïve mice in (A–D). The day 0 peritoneal MCp gated in (D) were from four naïve mice. The width of the photo in (C) corresponds to 64 μm. Color images available online at www.liebertpub.com/scd

Mentions: The Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp were cultured with IL-3 and stem cell factor to verify that the cells give rise to mast cells in vitro. After 5 days in culture, the MCp had proliferated and consisted of 100% c-kit+ FcɛRI+ mast cells (Fig. 3A). The cultured cells had a round nucleus and metachromatic granules after 14 days in culture, characteristics of mast cells (Fig. 3B). After 15 days in culture, 98.5% of the cells were c-kit+ FcɛRI+ mast cells (Fig. 3C). To confirm that MCp cultured in vitro (day 15) were more mature than primary MCp (day 0), the SSC light parameter was studied (Fig. 3D). The gating of the primary MCp was performed without the use of the SSC light parameter, that is, these primary cells were defined as Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi FcɛRI+ cells. After 15 days, the in vitro cultured MCp had a higher SSC light profile than the primary MCp (Fig. 3D).


Distinguishing Mast Cell Progenitors from Mature Mast Cells in Mice.

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

Doubly sorted Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp were cultured with IL-3 and stem cell factor. The cultured cells were analyzed after (A) 5 days, (B) 14 days, and (C, D) 15 days. Flow cytometric analysis was performed in (A, C, D), whereas May-Grünwald Giemsa staining was performed in (B). Primary MCp gated without the use of the SSC light profile (Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi FcɛRI+) were used as day 0 control in (D, black line). The cultured MCp were derived from six naïve mice in (A–D). The day 0 peritoneal MCp gated in (D) were from four naïve mice. The width of the photo in (C) corresponds to 64 μm. 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

f3: Doubly sorted Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp were cultured with IL-3 and stem cell factor. The cultured cells were analyzed after (A) 5 days, (B) 14 days, and (C, D) 15 days. Flow cytometric analysis was performed in (A, C, D), whereas May-Grünwald Giemsa staining was performed in (B). Primary MCp gated without the use of the SSC light profile (Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi FcɛRI+) were used as day 0 control in (D, black line). The cultured MCp were derived from six naïve mice in (A–D). The day 0 peritoneal MCp gated in (D) were from four naïve mice. The width of the photo in (C) corresponds to 64 μm. Color images available online at www.liebertpub.com/scd
Mentions: The Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp were cultured with IL-3 and stem cell factor to verify that the cells give rise to mast cells in vitro. After 5 days in culture, the MCp had proliferated and consisted of 100% c-kit+ FcɛRI+ mast cells (Fig. 3A). The cultured cells had a round nucleus and metachromatic granules after 14 days in culture, characteristics of mast cells (Fig. 3B). After 15 days in culture, 98.5% of the cells were c-kit+ FcɛRI+ mast cells (Fig. 3C). To confirm that MCp cultured in vitro (day 15) were more mature than primary MCp (day 0), the SSC light parameter was studied (Fig. 3D). The gating of the primary MCp was performed without the use of the SSC light parameter, that is, these primary cells were defined as Lin−/lo c-kithi T1/ST2+ CD16/32int integrin β7hi FcɛRI+ cells. After 15 days, the in vitro cultured MCp had a higher SSC light profile than the primary MCp (Fig. 3D).

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