Aequorin Expressed in Mitochondria Cells Human Umbilical Vein Endothelial Cells (HUVECs-AEQ-Mito)

Aequorin Expressed in Mitochondria Cells Human Umbilical Vein Endothelial Cells (HUVECs-AEQ-Mito)

Catalogue Number: UBP01AEQ

Product Format: Frozen Vial or T-25 flask

Cell Number: > 1x10^6 cells/vial

Description:
HUVECs are isolated from the normal human umbilical vein and HUVECs-AEQ-Mito cells are specifically selected from HUVECs infected with lentiviruses expressing Chimiral GFP-Aequorin in the mitochondria, using puromycin.

The cells are shipped in frozen vials (provided at passage 2). Universal Endothelial Growth Medium, containing 10% serum and growth supplements, is recommended for cell culture. These cells have a minimum average population doubling level of > 10 when cultured following the detailed protocol described below.

Characterization of the Cells:
1. Cytoplasmic VWF/Factor VIII >95% positive by immunofluorescence
2. Cytoplasmic uptake of DiI-Ac-LDL >95% positive by immunofluorescence
3. Cytoplasmic PECAM1 >95% positive by immunofluorescence
4. HUVECs are negative for HIV-1, HBV, HCV, and mycoplasma.

Product Usage:
Cells are offered for Research Use Only.

Shipping:
Frozen vials in a Dry Ice Package.

Handling of Arriving Cells:
Upon receiving the dry ice package with cells in frozen vials, transfer the vials into a -80°C freezer for short-term storage, or a liquid nitrogen tank for long-term storage.

Cell Culture Instructions From T-25 flask:

1. 
   1. Inspect to make sure the flask is at 90% confluence; if not, remove transport media and add 5ml of fresh media to the flask. Place the flask in a 37°C incubator until cells are at 90% confluence. Change media every 2 days.
   2. If the flask is at 90% confluence, aspirate transport media from the flask.
   3. Rinse the T-25 flask containing cells with 5 ml 1XPBS.
   4. Gently aspirate out the PBS after rinsing and discard.
   5. Add 2ml of room temperature Universal Detachment solution to the T-25 flask containing cells (ensure the entire interior surface is covered).
   6. Place the T-25 flask containing cells into a 37°C incubator for 1 or 2 minutes (cells will normally come off the surface within 1 or 2 minutes).
   7. Suspend the cells with 15ml of Universal ENDO-Growth medium and transfer equally into 3 pre-coated T-25 flasks (the cells are now at a subculture ratio of 1:3).
   8. There is no need to spin cells during subculture.
   9. Proliferating cells culture: Universal ENDO-Growth medium should be changed every 2 days. The cells normally become confluent within 7 days when split at a 1:3 ratio.
   10. Use ENDO-Basal media containing 0.5% FBS to induce quiescent cells (after 18-24 hours).

Welcome to the Thawing and Subculture Protocols to ensure optimal cell culture techniques and successful experiments.

A) Pre-coating of T-25 flasks:
Prepare the T-25 flask for cell culture by applying 2ml of Universal Coating Solution, ensuring full coverage. After 5 minutes, carefully remove any excess coating solution and rinse the flask twice with 5ml of 1x PBS. Your flask is now primed and ready for use.

B) Thawing the cells:
Thaw the frozen cell vial in a 37°C water bath, leaving a small amount of ice in the vial. Then, transfer the cells into the pre-coated T-25 flask with 10ml of growth medium. Usually, the cells will reach confluency overnight and be ready for passage within a few days.

C) Subculturing the cells:
Start by rinsing the cells in a T-25 flask with 5ml of room-temperature 1xPBS, followed by adding 2ml of room-temperature Universal Detachment Solution into the flask. Gently dispose of the excess solution within 20 seconds by aspiration.

D) Detaching the cells:
Leave the T-25 flask with the cells in a 37°C incubator for 1 minute. Most cells usually detach from the surface within 1-2 minutes. Alternatively, you can monitor the cells under a microscope until most of them become rounded up. Then gently tap the flask against the bench surface, and the cells will move on the surface of the flask when observed under a microscope.

E) Centrifugation:
Add 5ml of Universal Neutralization Buffer and spin down the cells with an 800g centrifugation for 5 minutes.

F) Re-suspending the cells:
Re-suspend the cell pellet with 10 or 15ml of Universal Growth Medium and transfer 5ml each into 2 or 3 pre-coated T25 flasks for a 1/2 to 1/3 subculture ratio.

G) Maintenance:
Ensure the medium is changed every 2 or 3 days, and the cells usually reach confluency within 7 days when split at a 1/3 ratio.

H) Preparation for experiments:
For experiments, to prepare quiescent cells, replace with Universal Endothelial Basal Medium containing 0.5% FBS when the cells are nearly confluent for about 8-12 hours before your experiments.

These protocols are designed to support the growth and health of your cells, ensuring the success of your important research.