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: Bone Research

Article Title: Endothelial PDGF-BB/PDGFR-β signaling promotes osteoarthritis by enhancing angiogenesis-dependent abnormal subchondral bone formation

doi: 10.1038/s41413-022-00229-6

Figure Lengend Snippet: PDGFR-β is primarily expressed in CD31 hi Emcn hi endothelial cells (ECs). a qRT‒PCR examination of PDGFR-β in CD31 hi Emcn hi ECs and CD31 lo Emcn lo ECs of bone marrow from normal mice. FACS analysis ( b ) and quantification ( c ) of CD31 hi Emcn hi ECs isolated from the bone marrow of the sham and 4-week-old and 8-week-old ACLT groups. n = 5 (5 mice with 10 subchondral bone specimens). d Confocal images (upper) with the magnified area (bottom) in the boxed area in the upper image of H-type vessels (endomucin: green; CD31: red; merge: yellow) and PDGFR-β + cells (white) of the sham and 4-week and 8-week post-ACLT groups. Scale bar, bottom 50 µm; top 100 µm. e , f Quantification of H-type vessel volume and PDGFR-β + cells in the sham and 4-week and 8-week post-ACLT groups. n = 10. Sham = sham controls. TV = total vessel volume; HV = H-type vessel volume. * P < 0.05 and ** P < 0.01 compared to the sham group or as denoted by bars

Article Snippet: FACS was conducted with antibodies against CD31-APC (RD, FAB3628A-025), CD45-Per-CP (BioLegend, 103133), Ter119-Brilliant Violet 421 (BioLegend, 116233), Endomucin-FITC (Santa Cruz, sc-65495), Sca-1-PE (BioLegend, 108107), and CD24-Brilliant Violet 421 (BioLegend, 101825).

Techniques: Isolation

Journal: Bone Research

Article Title: Endothelial PDGF-BB/PDGFR-β signaling promotes osteoarthritis by enhancing angiogenesis-dependent abnormal subchondral bone formation

doi: 10.1038/s41413-022-00229-6

Figure Lengend Snippet: Endothelial PDGFR-β regulates subchondral H-type vessels and the linked MSCs. FACS analysis ( a ) and quantification ( b ) of subchondral CD31 hi Emcn hi ECs isolated from the PDGFR-β lox/lox and PDGFR-β −/− mice at 4 weeks and 8 weeks post-ACLT. n = 5 (5 mice with 10 subchondral bone specimens). c Confocal images (upper) with the magnified area (bottom) in the boxed area in the upper image of H-type vessels (merge: yellow; endomucin: green; CD31: red) and LepR + cells (left part, white) and Nestin + cells (right part, white) in the subchondral bone from the PDGFR-β lox/lox and PDGFR-β −/− mice at 4 weeks post-ACLT. Scale bar, bottom 50 µm; top 100 µm. Quantification of subchondral H-type vessel volume ( d ), LepR + cells ( e ) and Nestin + cells ( f ) from the PDGFR-β lox/lox and PDGFR-β −/− mice at 4 weeks post-ACLT. n = 8. FACS ( g ) and quantification ( h ) of subchondral MSCs (CD45 − CD31 − Sca1 + CD24 + ) isolated from the PDGFR-β lox/lox and PDGFR-β −/− mice at 4 weeks and 8 weeks post-ACLT. μCT images of medial subchondral bone ( i ) and quantitative analysis of BV/TV ( j ), Tb.pf ( k ), SBP.Th ( l ), and Tb.N ( m ) in the subchondral bone from the PDGFR-β lox/lox and PDGFR-β −/− mice at 4 weeks and 8 weeks post-ACLT. n = 8. Scale bar, 500 µm. * P < 0.05 and ** P < 0.01 compared to the PDGFR-β lox/lox controls or as denoted by bars

Article Snippet: FACS was conducted with antibodies against CD31-APC (RD, FAB3628A-025), CD45-Per-CP (BioLegend, 103133), Ter119-Brilliant Violet 421 (BioLegend, 116233), Endomucin-FITC (Santa Cruz, sc-65495), Sca-1-PE (BioLegend, 108107), and CD24-Brilliant Violet 421 (BioLegend, 101825).

Techniques: Isolation

Journal: Bone Research

Article Title: Endothelial PDGF-BB/PDGFR-β signaling promotes osteoarthritis by enhancing angiogenesis-dependent abnormal subchondral bone formation

doi: 10.1038/s41413-022-00229-6

Figure Lengend Snippet: Specific inhibition of PDGFR-β in ECs attenuates OA development. a Confocal images (upper) with the magnified area (bottom) in the boxed area in the upper image of H-type vessels (merge: yellow; endomucin: green; CD31: red) and LepR + cells (left part, white) and Nestin + cells (right part, white) in the subchondral bone from the sham, ACLT + AAV control and ACLT + AAV rats at 8 weeks post-ACLT. Scale bar, bottom 50 µm; top 100 µm. Quantification of subchondral H-type vessel volume ( b ) and LepR + cells ( c ) and Nestin + cells ( d ) from the sham, ACLT + AAV control and ACLT + AAV rats at 8 weeks post-ACLT. n = 5. μCT images of medial subchondral bone ( e ) and quantitative analysis of BV/TV ( f ), Tb.pf ( g ), SBP.Th ( h ), and Tb.N ( i ) from the sham, ACLT + AAV control and ACLT + AAV rats at 8 weeks post-ACLT. n = 5. Scale bar, 500 µm. ( j ) SOFG (upper) with the magnified area (bottom) in the boxed area in the upper image of articular cartilage from the sham, ACLT + AAV control, and ACLT + AAV rats at 8 weeks post-ACLT; proteoglycan (red) and bone (green). Scale bar, bottom 50 µm; top 100 µm. k OARSI-modified Mankin scores of articular cartilage in sham, ACLT + AAV control, and ACLT + AAV rats at 8 weeks post-ACLT. n = 5. Sham = sham controls; ACLT + AAV control = AAV control-treated ACLT rats; ACLT + AAV = AAV for silencing endothelial PDGFR-β-treated ACLT rats; * P < 0.05 and ** P < 0.01 compared to the ACLT + AAV control group. # P < 0.05 and ## P < 0.01 compared to the sham group

Article Snippet: FACS was conducted with antibodies against CD31-APC (RD, FAB3628A-025), CD45-Per-CP (BioLegend, 103133), Ter119-Brilliant Violet 421 (BioLegend, 116233), Endomucin-FITC (Santa Cruz, sc-65495), Sca-1-PE (BioLegend, 108107), and CD24-Brilliant Violet 421 (BioLegend, 101825).

Techniques: Inhibition, Modification

Journal: Drug Design, Development and Therapy

Article Title: MK-4 Ameliorates Diabetic Osteoporosis in Angiogenesis-Dependent Bone Formation by Promoting Mitophagy in Endothelial Cells

doi: 10.2147/DDDT.S503930

Figure Lengend Snippet: MK-4 ameliorates the impairments of type H vessel formation and angiogenesis-dependent bone formation in DOP mice. ( a ) Schematic drawing of the procedures conducted in this study. ( b and c ) Representative flow cytometry plots of CD31 hi Emcn hi ECs (type H ECs). The bar graph showed the quantitation of type H ECs. ( d and e ) Confocal image of CD31 hi and Emcn hi (type H blood vessels) in mice models and histomorphometric quantitation of the area of type H blood vessels. Scale bar: 50μm. ( f and g ) Confocal image of Osterix + (green) osteoprogenitors around CD31 hi vessels (red) and the quantitative analysis. Scale bar: 50μm. ( h ) Typical three-dimensional and two-dimensional coronal images of the tibias were obtained by Micro-CT. ( i ) HE and Masson staining of mice tibias. Scale bar: 200μm or 100μm. ( j ) Statistical analysis of bone histological parameters. ( k and l ) IHC analysis of ALP and RUNX2 in the tibias of mice models. Scale bar: 50μm. Data were presented as mean ± SD (n=5). * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: The cells were subsequently washed and later incubated with PE-conjugated CD45 (E-AB-F1136D, 1:20; Elabscience, Wuhan, China) and APC-conjugated CD31 (E-AB-F1180E, 1:20; Elabscience) antibodies at 4 °C for 45 minutes.

Techniques: Flow Cytometry, Quantitation Assay, Micro-CT, Staining

Journal: Drug Design, Development and Therapy

Article Title: MK-4 Ameliorates Diabetic Osteoporosis in Angiogenesis-Dependent Bone Formation by Promoting Mitophagy in Endothelial Cells

doi: 10.2147/DDDT.S503930

Figure Lengend Snippet: 3-MA partially counteracts the promotion of type H vessel recovery and angiogenesis-dependent bone formation gain by MK-4. ( a and b ) Confocal image of CD31 hi and Emcn hi (termed type H blood vessels) in mice models and histomorphometric quantitation of the area of type H blood vessels. Scale bar: 50μm. ( c and d ) Confocal image of Osterix + (red) osteoprogenitors around Emcn hi vessels (green) and the quantitative analysis. Scale bar: 50μm. ( e ) HE and Masson staining of mice tibias. Scale bar: 200μm or 100μm. ( f ) Statistical analysis of bone histological parameters. ( g and h ) IHC analysis of ALP and RUNX2 in the tibias of mice models. Scale bar: 50μm. Data were presented as mean ± SD (n=5). * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: The cells were subsequently washed and later incubated with PE-conjugated CD45 (E-AB-F1136D, 1:20; Elabscience, Wuhan, China) and APC-conjugated CD31 (E-AB-F1180E, 1:20; Elabscience) antibodies at 4 °C for 45 minutes.

Techniques: Quantitation Assay, Staining