Thoracic aorta was obtained from 6-8 week male C57BL/6J mice after killing by CO2 asphyxiation and cervical dislocation in accordance with Schedule 1 Code of Practice, UK Animals Scientific Procedures Act 1986. The mice were also used for endothelial cell isolation from lungs by immunoselection with CD146 antibody–coated magnetic beads (Miltenyi Biotech, UK). Lungs were harvested in ice-cold Hanks’ balanced salt solution (HBSS), finely minced, and digested in HBSS containing 0.18 units.mL−1 collagenase (10 mg.mL−1; Worthington, USA) for 45 min at 37 °C. The digested tissue was filtered through a 70-μm cell strainer and centrifuged at 1000 rpm for 10 min. The cell pellet was washed with PBS/0.05% BSA, centrifuged, resuspended in 90 μL PBS/0.05% BSA, and incubated with 15 μL CD146 antibody-coated beads at 4 °C for 20 min. Bead-bound cells were separated from non-bead-bound cells using a magnet. Bead-bound cells (CD146 positive) were resuspended in 2 mL Endothelial Cell Medium MV2 (PromoCell), with manufacturer’s supplement, gentamicin, amphotericin-B, and 10 % FCS, and seeded onto fibronectin coated plates with a full media change at 2 and 24 h post-isolation. The cell population tested positive for the endothelial markers eNOS, Tie2, and CD102.
Cd146 antibody coated magnetic beads
Manufactured by Miltenyi Biotec
Sourced in United Kingdom
CD146 antibody–coated magnetic beads are a laboratory tool used for the isolation and enrichment of CD146-positive cells from a mixed cell population. The beads are coated with antibodies that specifically bind to the CD146 surface antigen, allowing for the separation and collection of CD146-expressing cells through magnetic separation techniques.
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2 protocols using cd146 antibody coated magnetic beads
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Isolation of Murine Lung Endothelial Cells
2
Isolation of Murine Lung Endothelial Cells
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Thoracic aorta was obtained from 6-8 week male C57BL/6J mice after killing by CO2 asphyxiation and cervical dislocation in accordance with Schedule 1 Code of Practice, UK Animals Scientific Procedures Act 1986. The mice were also used for endothelial cell isolation from lungs by immunoselection with CD146 antibody–coated magnetic beads (Miltenyi Biotech, UK). Lungs were harvested in ice-cold Hanks’ balanced salt solution (HBSS), finely minced, and digested in HBSS containing 0.18 units.mL−1 collagenase (10 mg.mL−1; Worthington, USA) for 45 min at 37 °C. The digested tissue was filtered through a 70-μm cell strainer and centrifuged at 1000 rpm for 10 min. The cell pellet was washed with PBS/0.05% BSA, centrifuged, resuspended in 90 μL PBS/0.05% BSA, and incubated with 15 μL CD146 antibody-coated beads at 4 °C for 20 min. Bead-bound cells were separated from non-bead-bound cells using a magnet. Bead-bound cells (CD146 positive) were resuspended in 2 mL Endothelial Cell Medium MV2 (PromoCell), with manufacturer’s supplement, gentamicin, amphotericin-B, and 10 % FCS, and seeded onto fibronectin coated plates with a full media change at 2 and 24 h post-isolation. The cell population tested positive for the endothelial markers eNOS, Tie2, and CD102.
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