Dear community,
Recently, I'm doing an experiment to measure the permeability of endothelial cells(HUVEC, HPAEC). I understand that it is important to make sure that endothelial cells form monolayer before proceeding with Permeability Assay. So, I'm going to dye the cell membrane of the vascular endothelial cells and check it with a fluorescence microscope. I'd like to ask you what reagents do you use to dye your cell membranes? What I found in my paper are the ones I suggested below, and I'm asking if it's okay to use to dye endothelial cells.
1. Dil Stain (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate ('DiI'; DiIC18(3))) (cat# D3911)
2. CellBriteTM Orange Cytoplasmic Membrane (cat# 30022)
3. CellMask™ Plasma Membrane Stains (cat#C10046)
Thanks for your attention.
I'd avoid lipophilic cyanine dyes, like DiI, because they label ALL membranes, internal organelles as well as PM. (they also don't survive fix and perm).
CellMask plasma membrane stains are excellent for PM and the most uniform choice for live cells. Another choice is Wheat Germ Agglutinin conjugates. They aren't quite as uniform, but they a better if you need to do fixation and permeabilization (if you need to perm, make sure to label FIRST before perming or they will also label internal organelles) and there are more color options. I did the testing and wrote the protocol for them when I was in R&D at Molecular Probes (now Thermo Fisher Scientific). CellMask PM stains lose some intensity with fixation and don't survive perm.
I've never used CellBrite, so I don't have an opinion on that one.
If you really want to be sure that you are working with endothelial cells, before proceeding with permeability assays, you can use von Willebrand factor which stains exclusively endothelial cells.
Best of luck.
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