HUVECs-GFP cell line熒光穩定表達細胞株

GENERAL INFORMATION
HUVECs were infected with GFP- expressing Lentiviral particles @ passage 1. Puromycin resistant GFP-HUVECs (cAP-0001GFP) were selected and shipped in frozen vials (cells are provided @ passage 3). Endothelial Growth Medium (cAP-02) is recommended for cell culture and these cells have a minimum of 18 population doubling levels when cultured following the protocols described below).
CHARACTERIZATION OF THE CELLS
1. Cytoplasmic VWF/Factor VIII >95% positive by immunofluorescence
2. Cytoplasmic uptake of Di-I-Ac-LDL >95% positive by immunofluorescence
3. Cytoplasmic PECAM1 >95% positive by immunofluorescence
4. HUVECs are negative for HIV-1, HBV, HCV, and mycoplasma

PROTOCOLS FOR THAWING THE CELLS AND SUBCULTURE
A) Pre-coating of T25 flasks- Add 2ml each Quick Coating Solution (cAP-01) into a T25 flask to cover the whole surface of the flask, 5 mins later, dispose the excessive coating solution by aspiration and the flask is ready to be used (although solution containing other extracellular matrix, i.e. gelatin, collagen, and fibronectin, can be used, make sure to optimize the conditions in advance).
B) Thaw the frozen cell vial in a 37C water bath first, and then transfer the cells into the pre-coated T25 flask with 10ml of cAP-02 medium, cells usually become confluent overnight and ready to be passaged.
C) To passage the cells, rinse the cells in a T25 flask with 5ml HBSS (RT) twice; then add 2ml Trypsin/EDTA (RT) (cAP-23) into one T25 flask; gently dispose the excessive Trypsin/EDTA solution within 20 seconds by aspiration.
D) Leave the T25 flask with the cells at RT or 37C for 1 min (most cells usually will detach from the surface within 1-2 mins; or monitor the cells under a microscope until most of cells become rounded up, and then gently tap the flask against the bench surface, and the cells will move on the surface of the flask when monitoring under microscope.
E) Add 5ml Trypsin Neutralization Buffer and spin down the cells with 800g centrifugation for 5 mins.
G) Re-suspend the cell pellet with 10 or 15ml cAP-2 medium and transfer 5 ml each into 2 or 3 pre-coated T25 flasks (for 1/2 to 1/3 subculture ratio).
H) Change medium every 2 or 3days and the cells usually become confluent within 7 days (when split at a 1/3 ratio).
I) To prepare quiescent cells, when cells are nearly confluent, replace cAP-02 with Endothelial Basal Medium (EBM, cAP-03) containing 0.5%FBS for about 8-12hrs before your experiments.

Supplier來源:BioVector NTCC Inc.
TEL電話:+86-010-53513060
Website網址: http://www.biovector.net