Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Reg1cp is involved in the regulation of CD31 hi EMCN hi endothelium formation. (A) Reg1cp expression levels in different cells isolated from human bone marrow. (B) Reg1cp expression levels in ECs. (C) The predicted secondary structure of Reg1cp and mutant Reg1cp around the mutant site (RNAfold Webserver, http://rna.tbi.univie.ac.at/cgibin/RNAfold.cgi ). (D) FACS analysis dot plot and quantitation of CD31 hi EMCN hi ECs (Type H ECs) of bone samples from one 37-yr-old male Reg1cp +/mut subject and five age-matched Reg1cp +/+ controls. (E) qRT-PCR analysis of CD31 and EMCN expression levels in ECs. (F) qRT-PCR analysis of VEGFA, VEGFB , PDGFA , PDFB , TGFβ , and FGF1 expression level in ECs. (G) Reg1cp expression level in human ECs transfected with Reg1cp-mut or Reg1cp-wt plasmid. (H and I) CD31 and EMCN expression levels (H) and VEGFA, VEGFB , PDGFA , PDFB , TGFβ , and FGF1 expression levels (I) in ECs. (J–L) Representative images of Alizarin Red S staining (J) and qRT-PCR analysis of the levels of SP7 and RUNX2 expression (K and L) in human BMSCs transfected with Reg1cp-mut or Reg1cp-wt plasmid with osteogenic induction. Scale bar, 0.5 cm. In A, B, E–I, K, and L, n = 5 in each group from three independent experiments. J is representative of three independent experiments. Data are shown as the mean ± SD. *, P < 0.05; **, P < 0.01; N.S, no significance; Student’s t test (B, E, and F) and ANOVA (A, G–I, K, and L). BMEPC, bone marrow endothelial progenitor cell; OB, osteoblast; PRE-OC, osteoclast precursor cell.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Expressing, Isolation, Mutagenesis, Quantitation Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Staining

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Mutant Reg1cp abolishes the role of KLF3 in CD31 hi EMCN hi vessel formation. (A) ChIP-PCR assays with anti-KLF3 antibodies or anti-IgG antibodies using specific primers targeting the promoter regions of JUNB . (B) qRT-PCR analysis of the JUNB expression level after anti-KLF3 or anti-IgG ChIP. (C) HMECs were transfected with luciferase reporter carrying WT-pGL3-JunB or MUT-pGL3-JunB, respectively, and cotransfected with the Klf3 plasmid or vector. Firefly luciferase values, normalized for renilla luciferase, are presented. (D and E) Western blotting analysis (D) and quantitation (E) of the relative levels of KLF3, JUNB, and VEGFA protein expression. (F) ChIP-PCR assays with anti-KLF3 antibodies or anti-IgG antibodies in HMECs transfected with Reg1cp-mut or Reg1cp-wt plasmids. (G) JUNB expression level of anti-KLF3 or anti-IgG ChIP. (H and I) Western blotting analysis (H) and quantitation (I) of the relative levels of KLF3, JUNB, and VEGFA protein expression. (J) ChIP-PCR assays with anti-KLF3 antibodies or anti-IgG antibodies in HMECs with different Reg1cp genotypes. Het , heterozygous mutation; Hom , homozygous mutation. (K) JUNB expression level of anti-KLF3 or anti-IgG ChIP. (L and M) Western blotting analysis (L) and quantitation (M) of the relative levels of JUNB and VEGFA protein expression. (N and O) Representative images (N) and relative quantification (O) of a transwell migration assay. Scale bar, 150 µm. (P and Q) Representative images (P) and relative quantification (Q) of tube branch numbers of a Matrigel tube formation assay. Scale bar, 750 µm. (R and S) HMECs were cultured under hypoxia for 24 h and analyzed (R) and quantified (S) for VEGFR2 phosphorylation (pVEGFR2, top) and VEGFR2 total levels (bottom). All panels were representative of three independent experiments. Data are shown as the mean ± SD. **, P < 0.01; Student’s t test (B, C, E, G, I, and K) and ANOVA (M, O, Q, and S).

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Mutagenesis, Quantitative RT-PCR, Expressing, Transfection, Luciferase, Plasmid Preparation, Western Blot, Quantitation Assay, Quantitative Proteomics, Transwell Migration Assay, Tube Formation Assay, Cell Culture, Phospho-proteomics

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Endothelial-specific Klf3 knockout mice show increased CD31 hi EMCN hi vessels and bone formation. (A) Expression level of Klf3 in ECs. (B) qRT-PCR analysis of JunB and Vegfa levels in ECs. (C and D) Representative images (C) and quantitation (D) of CD31 (green) and EMCN (red) immunostaining in femora from endothelial-specific Klf3 knockout mice ( Klf3 cdh5 −/− ) and their littermate controls ( Klf3 flox/flox ). Scale bars, 100 µm. (E and F) Quantitation (E) and FACS analysis dot plot (F) of CD31 hi EMCN hi ECs (Type H ECs) from long bones of 1-, 3-, and 12-mo-old Klf3 cdh5 −/− mice and their littermate controls. (G–K) Representative μCT images (G) and quantitative μCT analysis (H–K) of trabecular bone microarchitecture in femora. (L and M) Immunohistochemical staining (L) and quantification (M) of OCN + cells (brown) in femora. Scale bar, 50 µm. (N–P) Representative images of calcein double labeling of trabecular bone (N) with quantification of BFR per bone surface (BFR/BS; O) and MAR (P). Scale bar, 25 µm. (Q and R) Serum levels of OCN (Q) and CTX (R) at the time of harvest. n = 6 mice in each group from three independent experiments. Data are shown as the mean ± SD. *, P < 0.05; **, P < 0.01. Student’s t test.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Quantitation Assay, Immunostaining, Immunohistochemical staining, Staining, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Endothelial-specific Klf3 knockout in OVX mice show increased CD31 hi EMCN hi vessels and bone formation. (A and B) FACS analysis dot plot (A) and quantification (B) of CD31 hi EMCN hi ECs (Type H ECs). (C and D) Representative μCT images (C) and quantitative μCT analysis (D) of trabecular bone microarchitecture in femora. (E and F) Serum levels of OCN ( E) and CTX (F) at the time of harvest. (G and H) Representative images (G) and quantification (H) of ALP (green) immunostaining in femora. Scale bar, 200 µm. (I and J) Immunohistochemical staining (I) and quantification (J) of OCN + cells (brown) in femora. Scale bar, 50 µm. (K and L) Immunohistochemical staining (K) and quantification (L) of COL 1 (green) in femora. Scale bar, 200 µm. (M) Representative images of TRAP staining of femora. Scale bar, 50 µm. (N) Quantification data of TRAP + cells in trabecular bone surface. Number of TRAP + cells per bone perimeter (Tb.N.Trap + /B.Pm) was measured. n = 6 mice in each group from three independent experiments. Data are shown as the mean ± SD. **, P < 0.01; ANOVA.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Knock-Out, Immunostaining, Immunohistochemical staining, Staining

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: A natural compound is identified as a KLF3 inhibitor by molecular docking. (A–D) qRT-PCR analysis of the relative levels of CD31 (A), EMCN (B), JUNB (C), and VEGFA (D) expression in HMECs treated with four different compounds. n = 3 in each group from three independent experiments. (E) The structure of Ophiopogonin D selected by molecular docking. (F) Crystal structure of Ophiopogonin D bound to Klf3. (G) HPLC-MS chromatograms of Ophiopogonin D reference substance (upper panel) and KLF3 recruit ligand (lower panel). Representative of two independent experiments. (H) ChIP-PCR assays with anti-Klf3 antibodies or anti-IgG antibodies in HMECs treated with Ophiopogonin D and control groups. Representative of three independent experiments. (I) qRT-PCR analysis of JUNB expression after anti-KLF3 or anti-IgG ChIP. n = 3 in each group from three independent experiments. (J and K) Western blotting analysis (J) and the quantification (K) of the levels of JUNB and VEGFA in HMECs treated with vehicle or different doses of Ophiopogonin D. Representative of three independent experiments. Data are shown as the mean ± SD. *, P < 0.05; **, P < 0.01; Student’s t test (A–D and I) and ANOVA (K).

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Ophiopogonin D treatment promotes CD31 hi EMCN hi vessels and bone formation in aged mice. 12-mo-old C57/B6 mice were intraperitoneally treated with Ophiopogonin D at 20 mg/kg every other day for 3 mo. (A) qRT-PCR analysis of JunB level in ECs. (B) qRT-PCR analysis of Vegfa level in ECs. (C and D) FACS analysis dot plot (C) and quantification (D) of CD31 hi Emcn hi ECs (Type H ECs). (E and F) Representative images (E) and quantification (F) of CD31 (green) and EMCN (red) immunostaining in femora from Ophiopogonin D–treated mice and the vehicle control group. G, growth plate. B, bone. Scale bar, 100 µm. (G–K) Representative μCT images (G) and quantitative μCT analysis (H–K) of trabecular bone microarchitecture in femora. (L and M) Representative images (L) and quantification (M) of ALP (green) immunostaining in femora. Scale bar, 200 µm. (N and O) Immunohistochemical staining (N) and quantification (O) of OCN + cells (brown) in femora. Scale bar, 50 µm. (P and Q) Immunohistochemical staining (P) and quantification (Q) of COL 1 (green) in femora. Scale bar, 200 µm. (R and S) Serum levels of OCN (R) and CTX (S) at the time of harvest. (T–V) Representative images of calcein double labeling of trabecular bone (T) with quantification of BFR/BS (U) and MAR (V). Scale bar, 25 µm. n = 6 mice in each group from three independent experiments. Data are shown as the mean ± SD. *, P < 0.05; **, P < 0.01; ANOVA. Tb. BV/TV, trabecular bone volume per tissue volume; Tb. N, trabecular number; Tb. Sp, trabecular separation; Tb. Th, trabecular thickness.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Quantitative RT-PCR, Immunostaining, Control, Immunohistochemical staining, Staining, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Krüppel-like factor 3 inhibition by mutated lncRNA Reg1cp results in human high bone mass syndrome

doi: 10.1084/jem.20181554

Figure Lengend Snippet: Ophiopogonin D treatment promotes CD31 hi EMCN hi vessels and bone formation in OVX mice. 2-mo-old C57/B6 mice underwent OVX surgery and were intraperitoneally treated with Ophiopogonin D at 20 mg/kg every other day for 3 mo. (A and B) FACS analysis dot plot (A) and quantification (B) of CD31 hi EMCN hi ECs (Type H ECs). (C–G) Representative μCT images (C) and quantitative μCT analysis (D–G) of trabecular bone microarchitecture in femora. (H and I) Serum levels of OCN (H) and CTX (I) at the time of harvest. (J and K) Representative images (J) and quantification (K) of ALP (green) immunostaining in femora. Scale bar, 200 µm. (L and M) Immunohistochemical staining (L) and quantification (M) of COL 1 (green) in femora. Scale bar, 200 µm. (N and O) Immunohistochemical staining (N) and quantification (O) of OCN + cells (brown) in femora. Scale bar, 50 µm. (P) Representative images of TRAP staining of femora from Ophiopogonin D–treated mice and their controls. (Q) Quantification of TRAP + cells in trabecular bone surfaces. Number of TRAP + cells per bone perimeter (Tb.N.Trap + /B.Pm) was measured. Scale bar, 50 µm. ( n = 6 mice in each group from three independent experiments. Data are shown as the mean ± SD. **, P < 0.01; ANOVA (B and D–G) and Student’s t test (H, I, K, M, O, and Q). Tb. BV/TV, trabecular bone volume per tissue volume; Tb. N, trabecular number; Tb. Sp, trabecular separation; Tb Th, trabecular thickness.

Article Snippet: For mice samples, after filtration and washing, the cells were counted and incubated for 45 min at 4°C with EMCN antibody (1:100; SC-65495; Santa Cruz Biotechnology), then washed and further incubated with APC-conjugated CD31 antibody (1:100; FAB3628A; R&D Systems) for 45 min at 4°C.

Techniques: Immunostaining, Immunohistochemical staining, Staining

Journal: Journal of tissue engineering and regenerative medicine

Article Title: Portal venous repopulation of decellularised rat liver scaffolds with syngeneic bone marrow stem cells.

doi: 10.1002/term.3117

Figure Lengend Snippet: FIGURE 2 Flow cytometric analysis of cultured bone marrow for liver sinusoidal endothelial progenitor cells (CD133, CD45 and CD31), markers of endocytotic receptors (CD32, MRC and stabilin) and bone marrow mesenchymal stem cells (CD105, CD73 and CD90). Bone marrow was cultured for 5 days (n = 4) and used for flow cytometric analysis. (a) (i) Forward versus side scatter profile of cultured bone marrow cells, (ii) doublet exclusion and (iii) dead cell exclusion (negative for 7-AAD). (b) Gates were set according to live cultured unstained cells in the (i) PE, (ii) FITC and (iii) APC channels. (c) Samples were analysed in three antibody panels of three stains as follows: (i) CD133-PE CD45-FITC CD31-APC, (ii) CD32-PE MRCFITC stabilin-APC and (iii) CD105-PE CD73-FITC CD90-APC. Pseudocolour dot plots depicting expression of each cell surface marker in a representative sample of cultured bone marrow are shown. Figures within the dot plots represent percentage of parent population, that is, live cells. (d) Bar chart summarising expression of each cell surface marker as a percentage of live cells (error bars indicate standard error of the mean)

Article Snippet: Dead cells were routinely labelled with 7-aminoactinomycin D (7-AAD; BD Pharmingen) at 1 in 50 concentration for 30 min at 4 C in darkness, prior to incubation with fluorochromeconjugated antibodies for 30 min (FITC mouse antirat CD45, clone OX-1, Cat No. 561867, BD Pharmingen; PE mouse antirat FcγRIIb, clone D34-485, Cat No. 562189, BD Pharmingen; APC mouse antirat CD90, clone 0X-7, Cat No. 561409, BD Pharmingen; APC goat antirat CD31, Cat No. FAB3628A-025, R&D Systems; PE rabbit antimouse CD133, Cat No. NB120-16518PE, Novus Biologicals; FITC rabbit antirat mannose receptor, Cat No. bs-4727RFITC, Bioss Antibodies; Alexa-Fluor® 647 rabbit antirat stabilin-1, Cat No. bs-7510RA647, Bioss Antibodies; FITC rabbit antimouse CD73, Cat No. MBS2053160, MyBioSource; unconjugated mouse antirat endoglin, Cat No. sc-20072, Santa Cruz Biotechnology).

Techniques: Cell Culture, Expressing, Marker

Journal: Journal of tissue engineering and regenerative medicine

Article Title: Portal venous repopulation of decellularised rat liver scaffolds with syngeneic bone marrow stem cells.

doi: 10.1002/term.3117

Figure Lengend Snippet: FIGURE 6 Expression of CD31 and CD45 in native rat liver, rat bone marrow stem cells cultured in plastic culture flasks and rat bone marrow stem cells after 30 days in scaffold culture. CD31 and CD45 shown by (green) FITC-labelled secondary antibody, with nuclear DAPI labelling. (a) CD31 and (d) CD45 expression is seen in native rat liver. CD31 is absent in rat bone marrow stem cells when cultured in plastic flasks (b) (consistent with FACS expression profile shown in Figure 2) but present in the same cells in recellularised scaffolds (c). CD45 is present in rat bone marrow stem cells in culture (e) (consistent with FACS expression profile shown in Figure 2) but absent in the same cells in recellularised scaffolds (f)

Article Snippet: Dead cells were routinely labelled with 7-aminoactinomycin D (7-AAD; BD Pharmingen) at 1 in 50 concentration for 30 min at 4 C in darkness, prior to incubation with fluorochromeconjugated antibodies for 30 min (FITC mouse antirat CD45, clone OX-1, Cat No. 561867, BD Pharmingen; PE mouse antirat FcγRIIb, clone D34-485, Cat No. 562189, BD Pharmingen; APC mouse antirat CD90, clone 0X-7, Cat No. 561409, BD Pharmingen; APC goat antirat CD31, Cat No. FAB3628A-025, R&D Systems; PE rabbit antimouse CD133, Cat No. NB120-16518PE, Novus Biologicals; FITC rabbit antirat mannose receptor, Cat No. bs-4727RFITC, Bioss Antibodies; Alexa-Fluor® 647 rabbit antirat stabilin-1, Cat No. bs-7510RA647, Bioss Antibodies; FITC rabbit antimouse CD73, Cat No. MBS2053160, MyBioSource; unconjugated mouse antirat endoglin, Cat No. sc-20072, Santa Cruz Biotechnology).

Techniques: Expressing, Cell Culture

Journal: Carbohydrate polymers

Article Title: Highly elastic and bioactive bone biomimetic scaffolds based on platelet lysate and biomineralized cellulose nanocrystals.

doi: 10.1016/j.carbpol.2022.119638

Figure Lengend Snippet: Fig. 6. In vitro proangiogenic and chemotactic behavior of the cryogel scaffolds. Representative immunocytochemistry images of the (a–j) CD31 staining of the cryogel scaffolds seeded with hASCs and endothelial cells after 7 days of culture (blue: nuclei; red: CD31; green: cytoskeleton). Scale bar: 100 μm. (k) Directionality, (l) velocity, and (m) accumulated distance of the migrating endothelial cells in the presence of High m-CNCs content cryogel scaffolds releasates. Statistical sig nificance: *P < 0.05, and ****P < 0.0001.

Article Snippet: To evaluate the proangiogenic properties of the samples, they were processed as described in the above section and immunostained using a primary antibody anti CD31-APC conjugated (FAB3567A, R&D Systems, USA).

Techniques: In Vitro, Immunocytochemistry, Staining

Journal: ACS applied bio materials

Article Title: Hyaluronic Acid Oligomer Immobilization as an Angiogenic Trigger for the Neovascularization of TE Constructs.

doi: 10.1021/acsabm.1c00291

Figure Lengend Snippet: Figure 2. (A) DAPI (blue) and phaloidin (red) stained micrographs of HUVECs seeded onto GelMA hydrogels functionalized with A-HA formulations of LMW, HMW, or plain GelMA, cultured for 1, 3, 7, and 14 days. Scale bar measures 200 μm. (B) Confocal micrographs of HUVECs at day 7 stained with DAPI (blue) phalloidin (green) and immunolabeled samples with CD44 (yellow) and CD31 (red). Scale bar measures 100 μm. (C) dsDNA quantification and (D) metabolic activity normalized to dsDNA content for HUVECs seeded onto GelMA hydrogels, plain or functionalized with HMW or LMW A-HA, after 1, 3, 7, and 14 days. Tissue culture polystyrene was used as positive control (PC); N = 3. Statistical differences are represented in the graph for relevant parameters, with *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Samples were incubated with CD31 (Allophycocyanin/APC conjugated mouse monoclonal Anti-Human CD31/ PECAM-1, R&D Systems, U.S.A.), CD44 (PE Mouse Anti-Human CD44, BD Pharmingen, U.S.A.) or pERK1/2 (Mouse monoclonal to ERK1 + ERK2 (phospho T185 + Y187 + T202 + Y204), Abcam, U.K.) antibodies diluted in 0.1% Bovine Serum Albumin (BSA; SigmaAldrich, U.S.A.) in PBS overnight at −4 °C and under agitation.

Techniques: Staining, Cell Culture, Immunolabeling, Activity Assay, Positive Control

Journal: ACS applied bio materials

Article Title: Hyaluronic Acid Oligomer Immobilization as an Angiogenic Trigger for the Neovascularization of TE Constructs.

doi: 10.1021/acsabm.1c00291

Figure Lengend Snippet: Figure 3. (A) Representative micrographs of phosphorilated ERK1/2 in HUVECs seeded onto LMW, HMW, and GelMA, after 7 days in culture. (B) Gene expression analysis of HA binding and angiogenesis related markers CD44, KDR, CADH5, CD31, and RHAMM after 1,3 and 7 days of culture, with data presented for the LMW, HMW, GelMA, and Positive Control (PC) conditions. Normalization was performed to GAPDH expression in trypsinized HUVECs before seeding. ND (None detected) represents time points for which no template was amplified for a specific formulation. Statistical differences are represented in the graphs as *p < 0.05 and **p < 0.01.

Article Snippet: Samples were incubated with CD31 (Allophycocyanin/APC conjugated mouse monoclonal Anti-Human CD31/ PECAM-1, R&D Systems, U.S.A.), CD44 (PE Mouse Anti-Human CD44, BD Pharmingen, U.S.A.) or pERK1/2 (Mouse monoclonal to ERK1 + ERK2 (phospho T185 + Y187 + T202 + Y204), Abcam, U.K.) antibodies diluted in 0.1% Bovine Serum Albumin (BSA; SigmaAldrich, U.S.A.) in PBS overnight at −4 °C and under agitation.

Techniques: Gene Expression, Binding Assay, Positive Control, Expressing, Formulation

Journal: ACS applied bio materials

Article Title: Hyaluronic Acid Oligomer Immobilization as an Angiogenic Trigger for the Neovascularization of TE Constructs.

doi: 10.1021/acsabm.1c00291

Figure Lengend Snippet: Figure 5. (A) Representative three-dimensional projections of the hydrogels with cocultures of hDPCs and HUVECs after 7 days in culture. Surface view of the cell laden hydrogels immunolabeled with CD44 (yellow) and CD31 (red) and counterstained with DAPI (blue) and phalloidin (green). Side view projections of the cell-laden hydrogels, showing the distribution of (B) CD31 and (C) CD44 positive labeling. (D) Color quantification of cell markers as a function of the distance to the sample surface.

Article Snippet: Samples were incubated with CD31 (Allophycocyanin/APC conjugated mouse monoclonal Anti-Human CD31/ PECAM-1, R&D Systems, U.S.A.), CD44 (PE Mouse Anti-Human CD44, BD Pharmingen, U.S.A.) or pERK1/2 (Mouse monoclonal to ERK1 + ERK2 (phospho T185 + Y187 + T202 + Y204), Abcam, U.K.) antibodies diluted in 0.1% Bovine Serum Albumin (BSA; SigmaAldrich, U.S.A.) in PBS overnight at −4 °C and under agitation.

Techniques: Immunolabeling, Labeling