Human patient samples were collected at the Department of Neurosurgery of the University of Pennsylvania. The collection of human tissues, in compliance with the tissue banking protocol, was approved by the University of Pennsylvania Institutional Review Board, and written informed consent was obtained from each participant13 (link),14 (link). ECs were isolated and verified as previously described13 (link),14 (link). In brief, tumor-derived single-cell suspensions were prepared by the tissue bank. Cell suspensions were subjected to magnetic-activated cell sorting using anti-CD31 antibody-conjugated magnetic beads (1:100; Miltenyi Biotech; 130-091-935). Sorted ECs were verified by Dil-Ac-LDL (Alfa Aesar; J65597) absorption and von Willebrand factor staining.
Anti cd31 antibody conjugated magnetic beads
Manufactured by Miltenyi Biotec
Anti-CD31 antibody-conjugated magnetic beads are laboratory reagents designed to isolate and enrich cells expressing the CD31 surface marker. The beads are composed of a magnetic core coated with anti-CD31 antibodies, enabling the specific capture and separation of CD31-positive cells from a heterogeneous sample using a magnetic field.
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6 protocols using anti cd31 antibody conjugated magnetic beads
1
Isolation and Verification of Endothelial Cells from Tumor Samples
2
Isolation of Murine Aortic and Brain Endothelial Cells
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Mouse aortic ECs were isolated from Cdh5-CreERT2;Il6fl/fl mice as previously described35 (link). In brief, thoracic aorta was isolated from 3-week-old mice and cut into pieces. Aortic rings were embedded in phosphate-buffered saline (PBS)-prewashed Matrigel and then incubated in DMEM/F-12 medium containing 5% FBS for 5 days. After rinsing with PBS, the rings were removed and the remaining cells were incubated with 2 U/ml Dispase I (Gibco, 17105-041) for 20 min at 37 °C. After centrifugation at 500 × g for 10 min, the cell pellets were washed with PBS. ECs were also isolated from mouse brain. Single-cell suspension was prepared and subjected to MACS with anti-CD31 antibody-conjugated magnetic beads (Miltenyi Biotech, 130-091-935). Cells were cultured in DMEM/F-12 medium supplemented with 25 μg/ml EC growth supplement (Sigma) and 5% FBS. All cells were used between passages 2 and 4.
3
Isolation and Verification of Endothelial Cells
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Patient samples were collected at the Department of Neurosurgery of the Hospital of the University of Pennsylvania. The collection of human tissues in compliance with the tissue banking protocol was approved by the University of Pennsylvania Institutional Review Board, and written informed consent was obtained from each participant. ECs were isolated and verified14 (link). In brief, tumor-derived single-cell suspensions were prepared by the tissue bank. Red cells were removed with ACK lysis buffer (Life Technologies). Cell suspension was subjected to magnetic-activating cell sorting (MACS) with anti-CD31 antibody-conjugated magnetic beads (Miltenyi Biotech, 130-091-935). Sorted ECs were verified by Dil-Ac-LDL (Alfa Aesar, J65597) absorption, and 99% of cells were Dil-Ac-LDL-positive. All cells were checked and showed no mycoplasma contamination.
4
Isolation and Culture of GBM-Derived Endothelial Cells
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Surgical specimens from human patients with GBM ages 48 to 83 in all genders, multiple races, and ethnicities were collected at the Department of Neurosurgery of the Hospital of the University of Pennsylvania. The collection of human tissues in compliance with the tissue banking protocol was approved by the University of Pennsylvania Institutional Review Board, and written informed consent was obtained from each participant. ECs were isolated and verified as previously described (10 (link), 38 (link)). Tumor-derived single-cell suspensions were prepared by the tissue bank. Cell suspensions were subjected to magnetic-activated cell sorting (MACS) with anti-CD31 antibody-conjugated magnetic beads (Miltenyi Biotech, 130-091-935). All cells were used between passages 2 and 6. ECs were treated with harmine (10 to 40 μM; Sigma-Aldrich, 286044).
5
Isolation and Validation of GBM-Derived Endothelial Cells
6
Isolation of Tumor-Associated Endothelial Cells
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All patients received surgery at the Department of Neurosurgery of the University of Pennsylvania and were enrolled in a single institution tissue banking protocol that was approved by the University of Pennsylvania Human Studies Committee. Consent was obtained from all patients. Tumor-associated ECs were isolated as previously described66 (link). In brief, tumor-derived single-cell suspensions were prepared by the tissue bank. Red cells were removed with ACK lysis buffer (Life Technologies). Cell suspension was subjected to magnetic activating cell sorting (MACS) with anti-CD31 antibody-conjugated magnetic beads (Miltenyi Biotech, 130-091-935). Sorted ECs were verified by Dil-Ac-LDL (Bioquote) absorption, and over 99% of cells were Dil-Ac-LDL-positive.
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