Hello,
Please can some one help me.
We bought "Human Brain Microvascular Endothelial Cells" (HBMEC) cells including "Complete Classic Medium" culture medium, in the month of July 2017. We kept the cells in liquid nitrogen as soon as we received it .
I put the cells into culture on 01 August 2017, but they do not grow and do not attach either.
Here is the protocol used:
1- Preparation of the culture medium:
In a 500 ml bottle of Complete Classic Medium kit with serum, we added 10 ml of cultureBoost and 1 ml of Bac- Off, homogenize and make alliquots of 45ml, keep the alliquot at -20 ° C till the moment need of used;
2- Cell Culture:
- Preheat the attachment Factors at 37°C
- Completely coat 100-mm adherent cell plates with 1.5 ml preheat Attachment Factor
- Thaw the vial of HBMEC in 37°C water bath
- Culture the cells in the prepared plate in 10ml of Complet Classic Medium kit with Serum plus antibiotics
- Incubate the culture plate at 37°C in 5% CO2
- Add fresh medium daily for 6 days.
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