Pericyte progenitor cells were isolated from vein leftovers of patients undergoing coronary artery bypass graft surgery or varicose vein removal, as previously described 55 (link). In brief, saphenous veins were carefully dissected from surrounding tissues using a sterile scalpel and then thoroughly washed in PBS. Veins were manually minced prior to 4 hours incubation with 3.7 mg/mL Liberase 2 (Roche) and filtered passing the cell suspension through 30μm cell strainer. Cells were depleted for endothelial cells with anti-CD31 conjugated beads (Miltenyi), according to manufacturer’s instruction. The remaining cells were purified by selecting CD34+ cells by anti-CD34 beads (Miltenyi). Target cells were then plated on fibronectin (10 μg/mL) coated plates in presence of differentiation medium (EGM2-2% FBS, Lonza). Adherent colonies were passaged to new culture dishes once they reached 60-70% confluence and frozen stocks generated after Passage 2 (P2) for the experiments shown in this publication. Trypsin-EDTA (Life Technologies) was utilized to detach cells from the growth substrate.
Anti cd31 conjugated beads
Manufactured by Miltenyi Biotec
Sourced in United Kingdom
Anti-CD31 conjugated beads are a laboratory product used for the separation and isolation of cells expressing the CD31 cell surface marker. The beads are coated with anti-CD31 antibodies, which bind to the CD31 protein on the target cells, allowing their capture and isolation from a heterogeneous cell population.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd34 beads, by Miltenyi Biotec (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Liberase 2, by Roche (2 mentions)
Egm2 2 fbs, by Lonza (2 mentions)
Egm 2 medium, by Lonza (2 mentions)
Anti cd44, by Thermo Fisher Scientific (1 mentions)
Anti cd105, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Anti cd90, by BD (1 mentions)
4 protocols using anti cd31 conjugated beads
1
Isolation of Pericyte Progenitor Cells
2
Isolation of Pericyte Progenitor Cells
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Pericyte progenitor cells were isolated from vein leftovers of patients undergoing coronary artery bypass graft surgery or varicose vein removal, as previously described55 (link). In brief, saphenous veins were carefully dissected from surrounding tissues using a sterile scalpel and then thoroughly washed in PBS. Veins were manually minced before 4-h incubation with 3.7 mg ml−1 Liberase 2 (Roche) and filtered passing the cell suspension through a 30-μm cell strainer. Cells were depleted for ECs with anti-CD31-conjugated beads (Miltenyi), according to the manufacturer's instruction. The remaining cells were purified by selecting CD34+ cells by anti-CD34 beads (Miltenyi). Target cells were then plated on fibronectin (10 μg ml−1)-coated plates in presence of differentiation medium (EGM-2—2% FBS, Lonza). Adherent colonies were passaged to new culture dishes once they reached 60–70% confluence and frozen stocks generated after Passage 2 (P2) for the experiments shown in this publication. Trypsin-EDTA (Life Technologies) was utilized to detach cells from the growth substrate.
3
Isolation and Expansion of Cardiac Pericytes
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We have applied a good manufacturing practices-compliant standard operating procedure previously employed for derivation of pericytes from human saphenous vein.16 (link) CPs were isolated from atrium or ventricle specimens (3 to 5 mm, <100 mg) from infants and children undergoing surgical repair for congenital heart defects. In brief, discarded tissue was thoroughly washed in PBS and then manually minced. The tissue suspension was incubated for 40 minutes with 0.45 WU/mL/g Liberase 2 (Roche Technologies, UK). Passing the cell suspension sequentially through 70, 40, and 30-μm cell strainers ensured single cell suspension. Cells were depleted for ECs with anti-CD31 conjugated beads (Miltenyi Biotech, UK), following the manufacturer’s instructions. The remaining cells were purified by selecting CD34+ cells by anti-CD34 beads (Miltenyi Biotech). Target cells were cultured in the presence of EGM2 medium (Lonza, UK) supplemented with 2% fetal bovine serum (FBS). Adherent colonies were passaged to new culture dishes once they reached 60% to 70% confluence and frozen stocks were generated after Passage 2 for the experiments shown in this study. Trypsin-EDTA (Life Technologies, UK) was utilized to detach cells from the growth substrate.
4
Isolation and Culture of Saphenous Vein-Derived CD34+ Cells
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APCs were obtained from vein leftovers using a standard operating protocol described previously [21] . In brief, saphenous veins were carefully dissected from surrounding tissues using a sterile scalpel and then thoroughly washed in PBS. Veins were manually minced prior to 4 hour incubation with 3.7 mg/mL Liberase 2 (Roche Technologies). Single cell suspension was ensured by passing the dissociated material through a 30 μm cell strainer. The suspension was then depleted of endothelial cells using anti-CD31 conjugated beads (Miltenyi Biotech), followed by positive selection for CD34+ cells by anti-CD34 beads (Miltenyi Biotech). The immunosorted CD34+ cells were then plated on fibronectin (10 μg/mL) coated plates in presence of EGM2 medium (Lonza) supplemented with 2% FBS. Adherent colonies were passaged to new culture dishes once they reached 60-70% confluence.
All in vitro experiments were set up with cells at P6. Purity of the preparations was determined by flow cytometry using combinations of the following antibodies to confirm typical phenotype: anti-CD44 (ebioscience), anti-CD-105 (Life Technologies) and anti-CD90 (BD biosciences). After staining, fluorescence was analyzed using a FACS Canto II flow cytometer and FACS Diva software (both BD Biosciences). Flow-cytometry analysis indicates that >95% cells express the surface antigens CD105, CD44 and CD90.
All in vitro experiments were set up with cells at P6. Purity of the preparations was determined by flow cytometry using combinations of the following antibodies to confirm typical phenotype: anti-CD44 (ebioscience), anti-CD-105 (Life Technologies) and anti-CD90 (BD biosciences). After staining, fluorescence was analyzed using a FACS Canto II flow cytometer and FACS Diva software (both BD Biosciences). Flow-cytometry analysis indicates that >95% cells express the surface antigens CD105, CD44 and CD90.
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