Flow Cytometry In Mouse Primary Endothelial Cell Analysis

• General protocol for flow cytometry procedure to use an unconjugated primary antibody.
• Harvest Mouse Endothelial Cells and keep cells in the cell culture medium (M1168, freshly made Medium) containing 1-10 ng/ml VEGF and 10% FCS for 20 min at 37°C. (note: you may let cells over-growing for 24-48h after cells reach confluence; it may take 3-5 days for cells to reach confluence in a T25 flask)
• 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.
• You may let cells over-growing for 24-48h after cells reach confluence before doing any cell testing, cell staining, FACS or designed experiments.

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Uptake of Dil-Ac-LDL by Endothelial Cells

• Cells are seeded in a 24-well plate coated with gelatin-based coating solution (catalog # 6950, Cell Biologics) for 5-10 minutes to 70–90% confluence.
• Wash cells two times with 1% BSA in 1X PBS with Calcium & Magnesium (wash buffer).
• Incubate cells with cell culture medium containing 0.05% FBS for 2 hours in an incubator (37⁰C).
• Remove cell culture medium from the plate.
• Incubate endothelial cells with Dil-Ac-LDL for 4 h at 37°C. Note, mix Dil-Ac-LDL at the concentration of 10 ug/ml (Catalog No. L-35353/Alexa 594, Life Technologies) with complete cell culture medium containing 2-10 % FBS.
• Decant the medium and wash the cells 3 times with wash buffer, 3 min each wash.
• Keep the cells with 0.5-1.0 ml medium in the dark to analyze under a fluorescence microscope ASAP between 5 min - 4 hours (or fix cells and keep cells at 4°C in the dark until the scheduled time for analysis before 12-24 hours).
• For the best result, analyze the cells as soon as possible after staining procedure.

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Mouse Endothelial Tube Formation Assay (In Vitro Angiogenesis)

Preparing Matrigel:

• Thaw reduced growth factor matrigel (BD: 354230) by submerging the bottle on ice and store the matrigel at 4 °C overnight.

Preparing Cells:

• Wash 90-95% confluent endothelial cells with sterile PBS (1X) without calcium and magnesium twice.
• Incubate cells (on T25 or T75flask) with warm 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes at 37°C. Once cells detach (slightly taps on the flask can be used to speed up the detachment), add 6-10 ml culture medium (Cell Biologics' Cell Culture Medium supplemented with 5-10 % FBS) to the flask to end the digestion (neutralizing the Trypsin).
• Filter the cell suspension through a sterile 40µm nylon mesh filter (BD 352340) to remove remaining clumps.
• Count cells and resuspend cells to a final concentration of 2.5-5.0 x 10⁵ cells/ml in endothelial cell culture medium with 50 ng/ml VEGF.

Tube Formation Assay

• Pipet 200 µl matrigel into each well of a 24-well plate. Avoid creating bubbles.
• Incubate the 24-well plate at 37 °C and 5% CO₂ for 30 min to solidify matrigel.
• Once matrigel has set, seed 50,000 cells -100,000(200ul) into each well.
• Observe the tube formation after 4-6 hrs. Peak tube formation may occur between 12hrs and 24hrs. 
• Take image by 4X lenses microscope. 

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Protocol for immunofluorescence staining

·        Cells are seeded in a 24-well plate coated with gelatin-based coating solution (catalog # 6950, Cell Biologics) for 5-10 minutes to 90–100% confluence.

·        Wash cells two times with 1% BSA in 1X PBS with Calcium & Magnesium (wash buffer).

·        Fix cells 3.7% Formaldehyde (Dilute 37% stock in Blocking buffer) 20min at RT.

·        Remobilize cells (if need be) in Blocking buffer (0.4% Triton X 100, 1%BSA in 1xPBS) 20min at RT.

·        Add first antibody1:100-1:1000 in blocking buffer at 4⁰C overnight, and humidifier.

·        Wash the cells three times by using 1xPBS.

·        Second antibody 1:200 in blocking buffer, 1hr, RT, DARK, humidifier, and DAPI if needed.

·        Wash the cells three times by using 1xPBS.

·        Keep the cells with 0.5-1.0 ml 1xPBS in the dark to analyze under a fluorescence microscope or Mount with Prolong.

·        For the best result, analyze the cells as soon as possible after staining procedure.

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Note:

·        Adjust the volume of endothelial culture medium and number of cells plated depending on the type of endothelial cell used.

·        Please send us the cell images (>90% confluence) if you have any questions or problems with cultured cells.

Cell Biologics