HUVEC-GFP稳定表达细胞GFP Expressing Human Umbilical Vein Endothelial Cells

Name of Cells: GFP Expressing Human Umbilical Vein Endothelial Cells (GFP-

HUVECs)

Product Format: Proliferating cells in T25 or a Frozen Vial

Cell Number: > 90% confluent in T25 flask or Frozen Vial (>5x 105

/vial)

General Information

HUVECs  were isolated from normal human umbilical vein and transfected with GFP-Lentiviral

particles at passage one. Puromycin resistant GPF-HUVECs were selected and shipped in

proliferating culture with >90 confluence (the cells are provided @ passage 3). Endothelial Growth Medium

(EGM, contains 5% serum and growth supplements, cAP-02) is recommended for cell culture and these cells

have a minimum average population doubling levels > 18 when cultured following the detailed protocol

described below).

Representative images of GFP-HUVECs (Left panel:

Phase contrast image; Right panel: GFP image)

Handling of Arriving Cells

(1) When you receive the cells in a T25 flask, leave the T25 flask in 37°C CO2 incubator for 1 hour first,

and then replace the transport medium with fresh EGM full medium. Let the cells grow for 24 hour

before subculture.

(2) When you receive the cells in a frozen vial, you can transfer the vial of cells into a -80ºC freezer for

short period storage or a liquid nitrogen tank for long term storage. Thaw the cells in a 37°C water bath,

and then transfer the cells into a T25 flask pre-coated with Quick coating solution (cAP-01) as described

in details in Subculture Protocol.

Subculture Protocol

A) Pre-coating of T25 flasks: Add 2ml of Quick Coating Solution (cAP-01) into one T25 flask and make sure

whole surface of the flask is covered with the coating solution. Five minutes later, dispose excessive Quick

Coating Solution by aspiration and the flask is ready to be used (no need for overnight incubation when

using Quick Coating Solution). Other extracellular matrix can be used including gelatin, collagen, and

fibronectin and you are advised to test the conditions for using those materials in advance.

B) Rinse the cells in T25 flask with 5ml HBSS (Room Temperature, RT) twice.

C) Add 2ml of Trypsin/EDTA (RT) (cAP-23) into one T25 flask (make sure the whole surface of the T25 flask

is covered with Trypsin/EDTA), and gently dispose the excessive Trypsin/EDTA solution within 20

seconds with aspiration.

D) Leave the T25 flask with the cells at RT for 1 minute (the cells usually will detach from the surface within

1-2 minutes). You can monitor the cells under microscope and when most of cells become rounded up, hit

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 the cells down with 800g for 5 minutes.

F) Re-suspend the cell pellet with 10 - 20ml of EGM full medium and the cell suspension is transferred directly

into 2 or 4 pre-coated T25 flasks (5ml each, and the cells are sub-cultured at 1:2 to 1: 4 ratios)

G) Change medium every 2-3 days and cells usually become confluent within 7 days (when split at a 1:4 ratio).

H) If you need prepare quiescent cells, when cells are almost confluent, replace EGM full medium with

Endothelial Basal Medium (EBM, cAP-03) containing 0.5% FBS about 8-12 hours before your experiments.

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