The individual protocols are on their own pages:
- Flow Cytometry in Mouse Primary Endothelial Cell Analysis
- Uptake of Dil-Ac-LDL by Endothelial Cells
- Endothelial Tube Formation Assay (In Vitro Angiogenesis)
You may let cells over-grow for 24-48h after cells reach confluence before doing any cell testing, cell staining, FACS or designed experiments.
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.
· Permeabilize cells (if need be) in Blocking buffer (0.4% Triton X 100, 1%BSA in 1xPBS) 20min at RT.
· Add first antibody 1:100-1:1000 in blocking buffer at 4C 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
Imported from the previous cellbiologics.com site.
