Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 2. Ex vivo analysis of hPRL gene promoter activation. A, qPCR of hPRL promoter activity of PE , BM cells, and PBMC in T24 and S24 rats. A minimum of four samples from each treatment group was assayed in duplicate. Data are presented as mean SEM; *, P 0.05. B, Luciferase activity of PE and BM cells isolated from S24 and T24 rats. ***, P 0.001. C, Ex vivo bioluminescent imaging of FITC-labeled, CD11b adherent cells from the PE, BM, and PBMC of S24 transgenic rats. Scale bar, 50 m. D, Comparison of bioluminescence between PE cells from a S24 rat and a T24 rat. S24, saline 24 h; T24, TG 24 h; RLU, Relative luminescence units. Scale bar, 100 m.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Ex Vivo, Activation Assay, Activity Assay, Luciferase, Isolation, Imaging, Labeling, Transgenic Assay, Comparison, Saline

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 3. Identification of subpopulations of CD11b cells expressing luciferase in the PE. A, FACS analysis of cells isolated from PE of S24 rats: two-dimensional dot plot (ssc vs. fsc) showing two populations of CD11b-Alexa Fluor 647-stained cells (histograms, central panel). Blue, CD11b/sschigh; green, CD11B/ssclow. Diff-Quick staining of the corresponding sorted cell populations (right panels). Small black arrow, Mast cell, dark purple granules; small white arrow, eosinophils, red granules; large white arrow, B-cell, small cell with little cytoplasm; large black arrow, macrophage. Scale bar, 50 m. B, qPCR quantification of luciferase transcript in the S24 CD11b/ssclow population (n 5). C, FACS analysis of cells isolated from PE of T24 rats. Two-dimensional dot plot (ssc vs. fsc), representing three populations of CD11b-Alexa Fluor 647- and/or Gra-PE-stained cells (histograms, central panel). Blue, CD11b/sschigh; green, CD11b/Gra; pink, CD11b/Gra. Diff-Quick staining of the corresponding sorted cell populations (right panels). Scale bar, 50 m. D, qPCR quantification of luciferase transcript in the T24 CD11b/Gra (n 3) and CD11b/Gra (n 5) populations. E, RT-PCR for detection of extrapituitary luciferase and endogenous rPRL gene expression. In addition to the expected 122-bp product, a larger band (260 bp) was produced by alternative splicing as described previously (31). M, marker; 1, Luciferase from T24 CD11b/Gra; 2, rPRL from T24 CD11b/Gra; 3, Luciferase from T24 CD11b/Gra; 4, rPRL from T24 CD11b/Gra; 5, rPRL on pituitary (control); S24, saline 24 h; T24, TG 24 h.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Expressing, Luciferase, Isolation, Staining, Diff-Quik, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Produced, Alternative Splicing, Marker, Control, Saline

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 4. Subpopulations of CD11b cells expressing luciferase in BM and PBMC. A, FACS analysis of cells isolated from BM of T24 rats. Two- dimensional dot plot (ssc vs. fsc) showing two populations of CD11b-Alexa Fluor 647- and/or Gra-PE-stained cells (histograms, central panel). Blue, CD11b/sschigh (Gra); green, CD11b/ssclow (Gra). Diff-Quick staining of corresponding sorted cell populations (right panels). Black arrow, Monocytes; white arrow, neutrophils. Scale bar, 50 m. B, qPCR quantification of luciferase transcript in the S24 (n 5) and T24 (n 6) CD11b/ssclow population in BM. C, RT-PCR for the detection of extrapituitary luciferase and endogenous rPRL gene expression. M, Marker; 1 and 3, Luciferase on T24 CD11b/sschigh; 2 and 4, rPRL on T24 CD11b/sschigh; 5, rPRL on pituitary (control). D, FACS analysis of cells isolated from PB of T24 rats. Two-dimensional dot plot (ssc vs. fsc) showing two populations of CD11b-Alexa Fluor 647- and/or Gra-PE-stained cells (histograms, central panel). Blue, CD11b/Gra and green, CD11b/Gra. E, qPCR quantification of luciferase transcript in the S24 (n 5) and T24 (n 5) CD11b/Gra and T24 CD11b/Gra (n 5 in each group). S24, saline 24 h; T24, TG 24 h.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Expressing, Luciferase, Isolation, Staining, Diff-Quik, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Marker, Control, Saline

Journal: Endocrinology

Article Title: Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

doi: 10.1210/en.2011-1926

Figure Lengend Snippet: FIG. 7. Ex vivo stimulation of PRL expression in human peripheral blood monocytes. A, Peripheral blood monocytes stained with CD11b antibody (left panel). Diff-Quick stain of CD11b cells to confirm monocytes morphology. Scale bar, 50 m. B and C, RT-PCR analysis of two distinct donors, showing the bad pattern of extrapituitary hPRL gene expression. D and E, qPCR of hPRL extrapituitary gene expression in human CD11b PBMC of two donors. Cells were treated with TNF- (TNF), LPS, and TG for 16 h, then analyzed for hPRL gene expression. CycloA, Cyclophilin A; Ctrl, no-stimulation control.

Article Snippet: Adherent cells were blocked with a mouse antirat CD32 antibody (BD Pharmingen, San Diego, CA), then stained with an antirat CD11b fluorescein isothiocyanate (FITC)-conjugated antibody or matched isotype control (1:10 antibody; AbD Serotec, Kidlington, UK).

Techniques: Ex Vivo, Expressing, Staining, Diff-Quik, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control