Journal: PLoS ONE

Article Title: Fibrogenic Potential of Human Multipotent Mesenchymal Stromal Cells in Injured Liver

doi: 10.1371/journal.pone.0006657

Figure Lengend Snippet: A) Fibroblast-like morphology of adult (a) and pediatric (b) MSC. Both cell types showed similar morphology. B) Flow cytometry of cells stained for CD34, CD36, CD44, CD45, CD54, CD90, CD105, CD106, HLA ABC and isotype controls showing similar expression of surface antigens in adult (a) and pediatric (b) MSC. A representative example of adult and pediatric MSC is shown. Red histograms: specific marker; unfilled black histograms: isotype controls. C) Adipogenic differentiation was induced with adipocyte differentiation medium. After 3 weeks aMSC (a) and pMSC (b) were fixed and stained with Oil-red-O. In control medium (Insets), very few cells contained lipid droplets. In adipogenic differentiation medium a majority of cells contained lipid droplets stained in red. D) Chondrogenic differentiation was induced in MSC pellet culture in chondrogenic differentiation medium. After 4 weeks, pellets were fixed and sections were analyzed using Masson trichrome staining (a,b blue) and anti-collagen type II immunohistochemistry (c,d red). The images represent zoomed portion (delimitated by white frames) of the picture shown in the insets. The pellet structure appeared compact and contained abundant collagen fibrils. E) Osteogenic differentiation was induced by culturing aMSC (a) and pMSC (b) in osteogenic differentiation medium. After 4 weeks, cells were washed and incubated with Fast red and Sodium alpha-naphtyl phosphate solutions for 30 min at 4°C. Nuclei were stained with hemalun. MSC expressing alkaline phosphatase showed brown colored cytoplasm and displayed typical star-shaped cell morphology. In control medium (Insets), only few cells showed alkaline phosphatase expression.

Article Snippet: Following antibodies (Ab) were used for fluorescent activated cell sorting (FACS) analysis and immunohistochemistry: mouse anti-CD11b (Hycult biotechnology, Uden, the Netherlands), mouse anti-CD31 (Dako, Baar, Switzerland), phycoerythrin (PE)-conjugated mouse anti-CD34, Fluorescein isothiocyanate (FITC)-conjugated mouse anti-CD36, PE-mouse anti-CD44, PE-mouse anti-CD54, mouse anti-CD90, mouse anti-CD106, mouse isotype control (all from Becton Dickinson, Basel, Switzerland), PE-mouse anti-CD45 (R&D systems, Abingdon, UK), FITC-mouse anti-CD105 (Serotec, Oxford, UK), mouse anti-HLA-ABC (Chemicon Australia, Victoria, Australia), mouse anti-vimentin (Dako, Baar, Switzerland), mouse anti-human serum albumin, mouse anti-beta cytoplasmic actin (both from Sigma, Buchs, Switzerland), rabbit anti-human collagen type II (Mono-San, Uden, The Netherlands) and mouse anti-human alpha smooth muscle actin Ab .

Techniques: Flow Cytometry, Staining, Expressing, Marker, Immunohistochemistry, Incubation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

doi: 10.1038/s41374-018-0137-1

Figure Lengend Snippet: Antibody combinations used in immunophenotyping

Article Snippet: Fixation/Permeabilization Solution Kit with BD Golgi-PlugTM kit and Mouse Foxp3 Buffer Set was used for intracellular staining and purchased from BD Biosciences. table ft1 table-wrap mode="anchored" t5 caption a7 iMC CD11b Vio Blue Gr-1 APC-eFluor780 * Ly6b FITC Macrophages CD11b Vio Blue F4/80 APC CD68 PE mDC CD11b Vio Blue CD11c PE CD80 APC CD86 FITC pDC * CD11b Vio Blue CD11c PE B220 Vio770 Siglec H FITC CD4 T-cells CD3 APC-Cy7 CD4 eFluor450 CD4 T-regs CD3 APC-Cy7 CD4 eFluor450 CD25 PE FoxP3APC CD8 T-cells CD3 APC-Cy7 CD8 FITC B cells B220 Vio770 CD19 PE NK cells CD3 APC-Cy7 NKp46 FITC Neutrophils CD11b Vio Blue Gr-1 APC-eFluor780 Ly6b FITC * F4/80 APC Open in a separate window * negative tor this antibody Antibody combinations used in immunophenotyping table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Fluorochrome Vendor Clone Catalogue # Antibody Concentration (in 100 pL FACS buffer) CD3e APC-Cy7 Fisher Scientific 145–2C11 BDB557596 1 pL CD4 eFluor450 eBioscience GK1.5 48–0041-82 1 pL CD8 FITC eBioscience 53–6.7 11–0081-82 0.6 pL CD11b Vio Blue Miltenyi M1/70.15.11.5 130–097-336 3.75 pL CD11c PE Miltenyi N418 130102545 3.75 pL CD19 PE Miltenyi 6D5 130–102-598 5 pL CD25 PE eBioscience PC61.5 12–0251-81 0.5 pL CD68 PE Miltenyi FA-11 130–102-614 3.75 pL CD80 APC Miltenyi 16–10A1 130–102-584 3.75 pL CD86 FITC Miltenyi PO3.3 130–102-506 5 pL B220 APC-Vio770 Miltenyi RA3–6B2 130–102-267 3.75 pL F4/80 APC Miltenyi REA126 130–102-379 3.75 pL FoxP3 APC eBioscience FJK-16s 17–5773-82 5 pL Gr1 APC-eFluor780 eBioscience RB6–8C5 47–5931-80 1.2 pL Ly6b FITC abcam 7/4 ab53453 0.65 pL NKp46 FITC Miltenyi 29A1.4.9 130–102-300 3.75 pL SiglecH FITC eBioscience eBio440c 11–0333-82 0.5 pL Open in a separate window Details of antibodies used in the study

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

doi: 10.1038/s41374-018-0137-1

Figure Lengend Snippet: Details of antibodies used in the study

Article Snippet: Fixation/Permeabilization Solution Kit with BD Golgi-PlugTM kit and Mouse Foxp3 Buffer Set was used for intracellular staining and purchased from BD Biosciences. table ft1 table-wrap mode="anchored" t5 caption a7 iMC CD11b Vio Blue Gr-1 APC-eFluor780 * Ly6b FITC Macrophages CD11b Vio Blue F4/80 APC CD68 PE mDC CD11b Vio Blue CD11c PE CD80 APC CD86 FITC pDC * CD11b Vio Blue CD11c PE B220 Vio770 Siglec H FITC CD4 T-cells CD3 APC-Cy7 CD4 eFluor450 CD4 T-regs CD3 APC-Cy7 CD4 eFluor450 CD25 PE FoxP3APC CD8 T-cells CD3 APC-Cy7 CD8 FITC B cells B220 Vio770 CD19 PE NK cells CD3 APC-Cy7 NKp46 FITC Neutrophils CD11b Vio Blue Gr-1 APC-eFluor780 Ly6b FITC * F4/80 APC Open in a separate window * negative tor this antibody Antibody combinations used in immunophenotyping table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Fluorochrome Vendor Clone Catalogue # Antibody Concentration (in 100 pL FACS buffer) CD3e APC-Cy7 Fisher Scientific 145–2C11 BDB557596 1 pL CD4 eFluor450 eBioscience GK1.5 48–0041-82 1 pL CD8 FITC eBioscience 53–6.7 11–0081-82 0.6 pL CD11b Vio Blue Miltenyi M1/70.15.11.5 130–097-336 3.75 pL CD11c PE Miltenyi N418 130102545 3.75 pL CD19 PE Miltenyi 6D5 130–102-598 5 pL CD25 PE eBioscience PC61.5 12–0251-81 0.5 pL CD68 PE Miltenyi FA-11 130–102-614 3.75 pL CD80 APC Miltenyi 16–10A1 130–102-584 3.75 pL CD86 FITC Miltenyi PO3.3 130–102-506 5 pL B220 APC-Vio770 Miltenyi RA3–6B2 130–102-267 3.75 pL F4/80 APC Miltenyi REA126 130–102-379 3.75 pL FoxP3 APC eBioscience FJK-16s 17–5773-82 5 pL Gr1 APC-eFluor780 eBioscience RB6–8C5 47–5931-80 1.2 pL Ly6b FITC abcam 7/4 ab53453 0.65 pL NKp46 FITC Miltenyi 29A1.4.9 130–102-300 3.75 pL SiglecH FITC eBioscience eBio440c 11–0333-82 0.5 pL Open in a separate window Details of antibodies used in the study

Techniques: Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

doi: 10.1038/s41374-018-0137-1

Figure Lengend Snippet: Cell suspensions from BM and spleen were isolated from 8-week old mice. The cells were stained with B220 and CD19 antibodies for B cells, and CD3 and NKp46 antibodies for NK cells. The cells were then subjected to flow cytometry to determine cell-type percentages. (A) B cells in BM, (n≥10). (B) B cells in the spleen, (n ≥11). (C) NK cells in BM, (n≥9). (D) NK cells in the spleen, (n≥7). Results are shown as scatter plots depicting average cell percentages (percent of live cells). Error bars denote SEM. Each dot represents the value from a single mouse. (♂) and (♀) represent male and female mice respectively. **p ≤ 0.01, ***p ≤ 0.001, ****p≤0.0001.

Article Snippet: Fixation/Permeabilization Solution Kit with BD Golgi-PlugTM kit and Mouse Foxp3 Buffer Set was used for intracellular staining and purchased from BD Biosciences. table ft1 table-wrap mode="anchored" t5 caption a7 iMC CD11b Vio Blue Gr-1 APC-eFluor780 * Ly6b FITC Macrophages CD11b Vio Blue F4/80 APC CD68 PE mDC CD11b Vio Blue CD11c PE CD80 APC CD86 FITC pDC * CD11b Vio Blue CD11c PE B220 Vio770 Siglec H FITC CD4 T-cells CD3 APC-Cy7 CD4 eFluor450 CD4 T-regs CD3 APC-Cy7 CD4 eFluor450 CD25 PE FoxP3APC CD8 T-cells CD3 APC-Cy7 CD8 FITC B cells B220 Vio770 CD19 PE NK cells CD3 APC-Cy7 NKp46 FITC Neutrophils CD11b Vio Blue Gr-1 APC-eFluor780 Ly6b FITC * F4/80 APC Open in a separate window * negative tor this antibody Antibody combinations used in immunophenotyping table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Fluorochrome Vendor Clone Catalogue # Antibody Concentration (in 100 pL FACS buffer) CD3e APC-Cy7 Fisher Scientific 145–2C11 BDB557596 1 pL CD4 eFluor450 eBioscience GK1.5 48–0041-82 1 pL CD8 FITC eBioscience 53–6.7 11–0081-82 0.6 pL CD11b Vio Blue Miltenyi M1/70.15.11.5 130–097-336 3.75 pL CD11c PE Miltenyi N418 130102545 3.75 pL CD19 PE Miltenyi 6D5 130–102-598 5 pL CD25 PE eBioscience PC61.5 12–0251-81 0.5 pL CD68 PE Miltenyi FA-11 130–102-614 3.75 pL CD80 APC Miltenyi 16–10A1 130–102-584 3.75 pL CD86 FITC Miltenyi PO3.3 130–102-506 5 pL B220 APC-Vio770 Miltenyi RA3–6B2 130–102-267 3.75 pL F4/80 APC Miltenyi REA126 130–102-379 3.75 pL FoxP3 APC eBioscience FJK-16s 17–5773-82 5 pL Gr1 APC-eFluor780 eBioscience RB6–8C5 47–5931-80 1.2 pL Ly6b FITC abcam 7/4 ab53453 0.65 pL NKp46 FITC Miltenyi 29A1.4.9 130–102-300 3.75 pL SiglecH FITC eBioscience eBio440c 11–0333-82 0.5 pL Open in a separate window Details of antibodies used in the study

Techniques: Isolation, Staining, Flow Cytometry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

doi: 10.1038/s41374-018-0137-1

Figure Lengend Snippet: Cell suspensions from BM and spleen were isolated from 8-week old mice. The cells were stained with CD11c, B220 and Siglec H antibodies. CD11b antibody was included as a negative marker for pDCs. The cells were then subjected to flow cytometry to determine cell-type percentages. (A) pDCs in BM, (n≥11). (B) pDCs in the spleen, (n ≥9). Results are shown as scatter plots depicting average cell percentages (percent of live cells). Error bars denote SEM. Each dot represents the value from a single mouse. (♂) and (♀) represent male and female mice respectively.*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p≤0.0001.

Article Snippet: Fixation/Permeabilization Solution Kit with BD Golgi-PlugTM kit and Mouse Foxp3 Buffer Set was used for intracellular staining and purchased from BD Biosciences. table ft1 table-wrap mode="anchored" t5 caption a7 iMC CD11b Vio Blue Gr-1 APC-eFluor780 * Ly6b FITC Macrophages CD11b Vio Blue F4/80 APC CD68 PE mDC CD11b Vio Blue CD11c PE CD80 APC CD86 FITC pDC * CD11b Vio Blue CD11c PE B220 Vio770 Siglec H FITC CD4 T-cells CD3 APC-Cy7 CD4 eFluor450 CD4 T-regs CD3 APC-Cy7 CD4 eFluor450 CD25 PE FoxP3APC CD8 T-cells CD3 APC-Cy7 CD8 FITC B cells B220 Vio770 CD19 PE NK cells CD3 APC-Cy7 NKp46 FITC Neutrophils CD11b Vio Blue Gr-1 APC-eFluor780 Ly6b FITC * F4/80 APC Open in a separate window * negative tor this antibody Antibody combinations used in immunophenotyping table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Fluorochrome Vendor Clone Catalogue # Antibody Concentration (in 100 pL FACS buffer) CD3e APC-Cy7 Fisher Scientific 145–2C11 BDB557596 1 pL CD4 eFluor450 eBioscience GK1.5 48–0041-82 1 pL CD8 FITC eBioscience 53–6.7 11–0081-82 0.6 pL CD11b Vio Blue Miltenyi M1/70.15.11.5 130–097-336 3.75 pL CD11c PE Miltenyi N418 130102545 3.75 pL CD19 PE Miltenyi 6D5 130–102-598 5 pL CD25 PE eBioscience PC61.5 12–0251-81 0.5 pL CD68 PE Miltenyi FA-11 130–102-614 3.75 pL CD80 APC Miltenyi 16–10A1 130–102-584 3.75 pL CD86 FITC Miltenyi PO3.3 130–102-506 5 pL B220 APC-Vio770 Miltenyi RA3–6B2 130–102-267 3.75 pL F4/80 APC Miltenyi REA126 130–102-379 3.75 pL FoxP3 APC eBioscience FJK-16s 17–5773-82 5 pL Gr1 APC-eFluor780 eBioscience RB6–8C5 47–5931-80 1.2 pL Ly6b FITC abcam 7/4 ab53453 0.65 pL NKp46 FITC Miltenyi 29A1.4.9 130–102-300 3.75 pL SiglecH FITC eBioscience eBio440c 11–0333-82 0.5 pL Open in a separate window Details of antibodies used in the study

Techniques: Isolation, Staining, Marker, Flow Cytometry