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 cd34 beads
Manufactured by Miltenyi Biotec
Anti-CD34 beads are magnetic particles coated with antibodies against the CD34 cell surface antigen. These beads are used for the isolation and enrichment of CD34-positive cells from various sample sources, such as bone marrow, peripheral blood, and cord blood.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd31 conjugated 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 cd14 beads, by Miltenyi Biotec (1 mentions)
Diff quik, by Siemens (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
Nk cell isolation kit, by Miltenyi Biotec (1 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)
5 protocols using anti cd34 beads
1
Isolation of Pericyte Progenitor Cells
2
Purification of Immune Cell Subsets
3
Isolation of Pericyte Progenitor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
4
Isolation and Expansion of Cardiac Pericytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
5
Isolation and Culture of Saphenous Vein-Derived CD34+ Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!