Categories
Shopping Cart
InformationProtocol for Flow Cytometry, LDL uptake and Tube Formation Assay
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.
==================================================================
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.
==================================================================
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.
==================================================================
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.
==================================================================
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
