Flow Cytometry In Mouse Primary Endothelial Cell Analysis

General protocol for flow cytometry procedure to use an unconjugated primary antibody (2-step staining)

• Harvest Mouse Endothelial Cells (note: you may let cells over-growing for 24-48h after cells reach confluence; it may take 3-4 days for cell to reach confluence in a T25 flask) and keep cells in the cell culture medium (M1168, freshly made Medium, 1-2 weeks) containing 1-10 ng/ml VEGF and 10% FCS for 20 min at 37°C.
• Wash the cells 1 time with blocking buffer (1% BSA in 1X PBS with Calcium & Magnesium).
• Adjust Mouse Endothelial Cell suspension to a concentration of at least 0.5 x 10⁶ cells/ml in 1-2% BSA in 1X PBS with Calcium & Magnesium.
• Keep cells in blocking buffer for 30 min at RT.
• Add 0.1-10 μg/ml of the primary antibody (UNCONJUGATED). In general, we use 1.0 ug/ml of anti mouse CD31 antibody, Catalog No. AF3628, Anti-Mouse CD31/PECAM-1 (Polyclonal Goat IgG) from R&D SYSTEMS, INC. or CD31/PECAM-1 (Purified Rat Anti-Mouse CD31, Catalog No. 553370, BD).
• Take 1.0 ul primary antibody from stock solution (0.2mg/ml) into 200 ul sample.
• Incubate cells in eppendorf tubes for at least 30-45 min with gentle shaking at RT.
• Wash the cells two times (1-1.5 ml blocking buffer) by centrifugation at 200 g for 5 minutes and resuspend them in 500 µl.
• Add Second Antibody, Catalog No.: A-11055 (1:200 - 1:400 dilution, The Alexa Fluor® 488 Donkey Anti-Goat IgG (H+L), Invitrogen) and incubate in the dark at room temperature with gentle shaking for 30 minutes.
• Wash the cells 2 times (1-1.5 ml blocking buffer) by centrifugation at 200 g for 5 minutes and resuspend them in 0.4 ml (5 ml Round-Bottom Tube with Cell-Strainer Cap, Catalog No. 352235 BD).
• Keep the cells in the dark on ice and analyze the cells ASAP between 5 min - 2 hours by FACS.