Journal: Journal of Cellular and Molecular Medicine

Article Title: Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration

doi: 10.1111/j.1582-4934.2010.01087.x

Figure Lengend Snippet: Flow cytometry analysis of the cell population obtained by peritoneal lavage in control unoperated mice, 24 and 48 hrs after injury of the peritoneal wall. Representative result from a series of experiments described in the text. Top row: Isotype control (rat IgG2a) versus GFP. About two thirds of the cells present in the peritoneal lavage are GFP + . Second row: Mesothelin versus GFP. In the unoperated mouse, 4.2% of the cells are mesothelin + /GFP – . They probably are delaminated mesothelial cells. Because these cells can be considered as a positive control of the antibody, we used this population to establish the limit between the mesothelin + and mesothelin – cells. According to this criterion, 16.6% and 6.3% of the cells were mesothelin + /GFP + by 24 and 48 hrs after surgery. Note the increase of mesothelin + /GFP – cells by 24 hrs, reaching more than 10% of the total population. Bottom rows: Gating on a F4/80 + /GFP + population obtained from peritoneal lavages originates similar SS/FS profiles from unoperated and operated mice. However, gating on the mesothelin + /GFP + population originates a different profile, suggesting that both populations are different.

Article Snippet: The primary antibodies used were: rat antimouse CD45, PE conjugated (Pharmigen, Becton Dickinson, Franklin Lakes, NJ, USA, Clone 30-F11) diluted 1:500; rat antimouse mesothelin (MBL D053, clone 295D; MBL, Woburn, MA, USA) diluted 1:50; rat antimouse F4/80, FITC conjugated (eBioscience 11–4801, Clone BM8; eBioscience, San Diego, CA, USA) diluted 1:100.

Techniques: Flow Cytometry, Positive Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration

doi: 10.1111/j.1582-4934.2010.01087.x

Figure Lengend Snippet: The CD45 + fraction of the peritoneal lavage cells obtained 48 hrs after injury of the peritoneal wall and purified by magnetic immunobeads, shows mesothelin mRNA expression by RT-PCR. Positive control (C+) consisted of mesothelial cells obtained by mild trypsinization of the peritoneal cavity. Negative control (C–) was performed with free peritoneal cells obtained from a mesothelin-null mouse. Normalization of RT-PCR product was made with expression of β-actin as reference gene.

Article Snippet: The primary antibodies used were: rat antimouse CD45, PE conjugated (Pharmigen, Becton Dickinson, Franklin Lakes, NJ, USA, Clone 30-F11) diluted 1:500; rat antimouse mesothelin (MBL D053, clone 295D; MBL, Woburn, MA, USA) diluted 1:50; rat antimouse F4/80, FITC conjugated (eBioscience 11–4801, Clone BM8; eBioscience, San Diego, CA, USA) diluted 1:100.

Techniques: Purification, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration

doi: 10.1111/j.1582-4934.2010.01087.x

Figure Lengend Snippet: Antigen immunolocalization in adherent cells obtained from peritoneal lavages 48 hrs after injury of the peritoneal wall. (A–C) show cells obtained from normal BALB/c mice whereas (D)–(F) show cells from mice reconstituted with GFP-expressing bone marrow. (A) Virtually all the CD45 + cells show a perinuclear dot-like pattern of cytokeratin immunoreactivity. Note a strongly CD45 – /cytokeratin + immunoreactive cell, probably a delaminated mesothelial cell (arrow). (B) The perinuclear dot-like pattern of cytokeratin immunoreactivity is also present in mesothelial cells migrating from omentum explants, which were used as positive controls (arrows). Other cells still show an extended cytokeratin cytoskeleton (arrowhead). (C) Mesothelin immunoreactivity colocalized with CD68. Note the cytoplasmic and perinuclear CD68 localization. (D) Triple localization of mesothelin, cytokeratin and GFP. This immunostaining revealed different phenotypes, including triple positive cells (arrowheads), mesothelin + /cytokeratin + , GFP – cells which probably are delaminated mesothelial cells (white arrows) and GFP + , mesothelin – /cytokeratin – cells (yellow arrow). (E) The fibroblast marker FSP1 was expressed in many GFP + cells (white arrows), as well as in GFP – cells (yellow arrows). Other GFP + cells were negative for FSP1 (arrowhead). (F) Negative control incubated with isotype primary antibodies and with the same secondary antibodies as used in the rest of the figures.

Article Snippet: The primary antibodies used were: rat antimouse CD45, PE conjugated (Pharmigen, Becton Dickinson, Franklin Lakes, NJ, USA, Clone 30-F11) diluted 1:500; rat antimouse mesothelin (MBL D053, clone 295D; MBL, Woburn, MA, USA) diluted 1:50; rat antimouse F4/80, FITC conjugated (eBioscience 11–4801, Clone BM8; eBioscience, San Diego, CA, USA) diluted 1:100.

Techniques: Expressing, Immunostaining, Marker, Negative Control, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration

doi: 10.1111/j.1582-4934.2010.01087.x

Figure Lengend Snippet: Colocalization of GFP and mesothelin, cytokeratin, F4/80, FSP1 and procollagen-1 in the injured (INJ) or contralateral (CL) peritoneal wall from mice reconstituted with GFP-expressing bone marrow, 48 hrs after surgery. (A–D) Colocalization of GFP and mesothelin in the contralateral areas. Some double-labelled cells are apparently detaching from the mesothelial surface (arrows). GFP + cells within the tissue are mesothelin – as shown in (B). GFP + /mesothelin – cells are also abundant in sub-mesothelial areas (arrowheads). (E) Double labelled cells can also be seen in the regenerating mesothelium of the injured surface (arrows). GFP + /mesothelin – cells are also present but they are less abundant than in contralateral areas (arrowhead). (F), (G) Colocalization of GFP and cytokeratin can be observed in the contralateral areas (arrows in F) but it is apparently scarcer in the injured ones (G). GFP + /cytokeratin – cells are shown in the contralateral side (arrowheads in F). (H), (I) The macrophage marker F4/80 is present in a few cells from both areas (arrows). However, most of the apparently adhered GFP + cells in the injured area do not express this macrophage marker (arrowheads). (J), (K) Colocalization of GFP with the fibroblastic marker FSP1 (arrows). (L), (M) Colocalization of GFP with procollagen-1 (arrows).

Article Snippet: The primary antibodies used were: rat antimouse CD45, PE conjugated (Pharmigen, Becton Dickinson, Franklin Lakes, NJ, USA, Clone 30-F11) diluted 1:500; rat antimouse mesothelin (MBL D053, clone 295D; MBL, Woburn, MA, USA) diluted 1:50; rat antimouse F4/80, FITC conjugated (eBioscience 11–4801, Clone BM8; eBioscience, San Diego, CA, USA) diluted 1:100.

Techniques: Expressing, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration

doi: 10.1111/j.1582-4934.2010.01087.x

Figure Lengend Snippet: Colocalization of GFP, mesothelial and fibroblastic markers in the contralateral (CL) and injured (INJ) peritoneal wall from mice reconstituted with GFP-expressing bone marrow, 1 week (A–C) and 1 month (D–F) after surgery. (A) Colocalization of cytokeratin and GFP in the injured area 1 week after surgery. Some GFP + cells show an extended cytokeratin cytoskeleton (arrowhead) whereas others show the perinuclear dot-like cytokeratin pattern (arrows). (B), (C) Colocalization of smooth muscle cell α-actin with GFP in the injured area 1 week after surgery. GFP + cells are very abundant and form several cell layers covering all the damaged area. Most of these cells express smooth muscle cell α-actin. (D–F) Colocalization of mesothelial markers and GFP in the injured and contralateral areas 1 month after surgery. The mesothelium is completely regenerated. GFP + cells are present in the sub-mesothelial area, and some of them show a perinuclear cytokeratin dot (arrows in D’). It is still possible to find a few GFP + cells integrated in the mesothelial lining of both, injured and contralateral areas, expressing cytokeratin (arrows in E) and mesothelin (arrows in F).

Article Snippet: The primary antibodies used were: rat antimouse CD45, PE conjugated (Pharmigen, Becton Dickinson, Franklin Lakes, NJ, USA, Clone 30-F11) diluted 1:500; rat antimouse mesothelin (MBL D053, clone 295D; MBL, Woburn, MA, USA) diluted 1:50; rat antimouse F4/80, FITC conjugated (eBioscience 11–4801, Clone BM8; eBioscience, San Diego, CA, USA) diluted 1:100.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration

doi: 10.1111/j.1582-4934.2010.01087.x

Figure Lengend Snippet: Expression of markers on macrophages, PRC, fibrocytes and fibroblasts according our own results and data from [ 23 ]

Article Snippet: The primary antibodies used were: rat antimouse CD45, PE conjugated (Pharmigen, Becton Dickinson, Franklin Lakes, NJ, USA, Clone 30-F11) diluted 1:500; rat antimouse mesothelin (MBL D053, clone 295D; MBL, Woburn, MA, USA) diluted 1:50; rat antimouse F4/80, FITC conjugated (eBioscience 11–4801, Clone BM8; eBioscience, San Diego, CA, USA) diluted 1:100.

Techniques: Expressing