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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) Peritoneal lavage cells were analyzed with flow cytometry. ILC2s were gated as Lin− c-kit−/lo FcɛRI− CD127+ CD25+ T1/ST2+ CD16/32− cells, MCp as Lin−/lo c-kithi FcɛRI+ CD127− CD25− T1/ST2+ CD16/32int SSClo integrin β7hi cells, and mature mast cells (MC) as Lin−/lo c-kithi FcɛRI+ CD127− CD25− T1/ST2+ CD16/32int SSChi integrin β7−/lo/int cells. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the total number of peritoneal cells. (B) The ILC2s, mature mast cells, and MCp were doubly sorted. One hundred cells of each population were isolated, and a gene expression microarray analysis was performed. The gene expression profiles of ILC2s, mature mast cells, and MCp are shown as a heat map. The scale shows the log2-fold change in gene expression. Each respective population was sorted from peritoneal lavage cells pooled from five naïve mice. ILC2s, innate lymphoid cells of type 2. Color images available online at www.liebertpub.com/scd
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f4: (A) Peritoneal lavage cells were analyzed with flow cytometry. ILC2s were gated as Lin− c-kit−/lo FcɛRI− CD127+ CD25+ T1/ST2+ CD16/32− cells, MCp as Lin−/lo c-kithi FcɛRI+ CD127− CD25− T1/ST2+ CD16/32int SSClo integrin β7hi cells, and mature mast cells (MC) as Lin−/lo c-kithi FcɛRI+ CD127− CD25− T1/ST2+ CD16/32int SSChi integrin β7−/lo/int cells. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the total number of peritoneal cells. (B) The ILC2s, mature mast cells, and MCp were doubly sorted. One hundred cells of each population were isolated, and a gene expression microarray analysis was performed. The gene expression profiles of ILC2s, mature mast cells, and MCp are shown as a heat map. The scale shows the log2-fold change in gene expression. Each respective population was sorted from peritoneal lavage cells pooled from five naïve mice. ILC2s, innate lymphoid cells of type 2. Color images available online at www.liebertpub.com/scd

Mentions: The log2-fold difference in gene expression is shown in Fig. 4B. Heat maps were generated using Genesis 1.7.6 [8]. The data were adjusted by mean centering the gene expression. The gene expression data have been deposited in NCBI's Gene Expression Omnibus, and they are publicly accessible through the GEO Series accession number GSE63507 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63507).


Distinguishing Mast Cell Progenitors from Mature Mast Cells in Mice.

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

(A) Peritoneal lavage cells were analyzed with flow cytometry. ILC2s were gated as Lin− c-kit−/lo FcɛRI− CD127+ CD25+ T1/ST2+ CD16/32− cells, MCp as Lin−/lo c-kithi FcɛRI+ CD127− CD25− T1/ST2+ CD16/32int SSClo integrin β7hi cells, and mature mast cells (MC) as Lin−/lo c-kithi FcɛRI+ CD127− CD25− T1/ST2+ CD16/32int SSChi integrin β7−/lo/int cells. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the total number of peritoneal cells. (B) The ILC2s, mature mast cells, and MCp were doubly sorted. One hundred cells of each population were isolated, and a gene expression microarray analysis was performed. The gene expression profiles of ILC2s, mature mast cells, and MCp are shown as a heat map. The scale shows the log2-fold change in gene expression. Each respective population was sorted from peritoneal lavage cells pooled from five naïve mice. ILC2s, innate lymphoid cells of type 2. 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

f4: (A) Peritoneal lavage cells were analyzed with flow cytometry. ILC2s were gated as Lin− c-kit−/lo FcɛRI− CD127+ CD25+ T1/ST2+ CD16/32− cells, MCp as Lin−/lo c-kithi FcɛRI+ CD127− CD25− T1/ST2+ CD16/32int SSClo integrin β7hi cells, and mature mast cells (MC) as Lin−/lo c-kithi FcɛRI+ CD127− CD25− T1/ST2+ CD16/32int SSChi integrin β7−/lo/int cells. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the total number of peritoneal cells. (B) The ILC2s, mature mast cells, and MCp were doubly sorted. One hundred cells of each population were isolated, and a gene expression microarray analysis was performed. The gene expression profiles of ILC2s, mature mast cells, and MCp are shown as a heat map. The scale shows the log2-fold change in gene expression. Each respective population was sorted from peritoneal lavage cells pooled from five naïve mice. ILC2s, innate lymphoid cells of type 2. Color images available online at www.liebertpub.com/scd
Mentions: The log2-fold difference in gene expression is shown in Fig. 4B. Heat maps were generated using Genesis 1.7.6 [8]. The data were adjusted by mean centering the gene expression. The gene expression data have been deposited in NCBI's Gene Expression Omnibus, and they are publicly accessible through the GEO Series accession number GSE63507 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63507).

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