Hello. My team has just revived bEnd.3 yesterday from a cryovial stored in liq nitrogen, purchased from ATCC, following the protocol stated by ATCC. After 24 hours, we only saw about 1-2 spindle shaped endothelial-like cells. The other cells look rounded, not fully adhering. If you have any experience with bEnd.3, could you please kindly share your experience when you were reviving your cells. When should I change the medium? How long should I leave it before I passage them? Thanks.
HI! bEnd do tend to look roundish when not stimulated, and spindle-like when stimulated... Could it be that you're using to little supplements? Try adding 0,1mg/ml heparin and 0,05mg/ml Endothelial Cell Growth Supplement (ECGS), both from sigma. We add this directly to the culture flask rather than the medium bottle, somehow that seems better... Medium change every 2-3days. Sometimes increasing FBS to 20% also helps, but 10% should be enough.
Also, from my experience, I have a feeling that F12 (or F12:DMEM at 1:1) is preferable for endothelial cells than DMEM...
Hi Helder. I currently do not have 0,1mg/ml heparin and 0,05mg/ml Endothelial Cell Growth Supplement (ECGS) at the moment, so I will opt to add 20% FBS for the time being. Let's pray that this works!
Hi! if you have access to positively-charged plastic, it might help also...
Keep in mind that 20% FBS is quite high, plan your experiments in advance so you can reduce FBS to 1% an overnight before the proper experiment.
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