Human Coronary Artery Endothelial Cells

We are excited to present vital information about our outstanding product:

Product Details:
- Product Name: Human Coronary Artery Endothelial Cells
- Catalog Number: UBP07
- Storage: It is recommended to store at 37°C in a CO2 incubator for optimal conditions.
- Product Format: These high-quality cells are available as proliferating culture or in a conveniently frozen state.
- Cell Numbers: You can expect cells to be >90% confluent in a T25 flask or find 500,000 cells in a frozen vial.

*Caution: Handling human-derived products carries potential biological hazards. Rest assured, all cell strains have been meticulously tested negative for HIV, HBV, and HCV DNA in diagnostic tests. To maintain a safe working environment, always ensure proper protective equipment, such as gloves and safety glasses, are worn. We strongly advise following universal procedures for handling products of human origin as the minimum precaution against contamination.

General Information:
- Our Human Coronary Artery Endothelial Cells have been meticulously isolated from normal human coronary arteries. Passage 1 cells are conveniently shipped frozen in a 1ml vial or in a T-25 Flask with a confluence of >90%. For successful culturing, we recommend using the ENDO-Growth medium containing 5% serum and growth supplement. These exceptional cells have an average population doubling level of >16 when cultured. Upon receipt of the cells, it is advised to leave the flask in a 37°C CO2 incubator for 1 hour. Following this, replace the transport medium with fresh ENDO-Growth medium. Allow the cells to expand for 24 hours before subculture.

Cell Characterization:
- Through immunofluorescence, it is evident that Cytoplasmic VWF/factor VIII, Cytoplasmic uptake of Di-I-Ac-LDL, and Cytoplasmic PECAM1 are all >95% positive. Additionally, our Human Coronary Artery Endothelial Cells have been rigorously tested and are confirmed to be negative for HIV-1, HBV, HCV, and mycoplasma.

Product Use and Shipping Information:
- Emphasizing that our Human Coronary Artery Endothelial Cells are strictly for research use. They will be efficiently shipped as a proliferating culture in a T25 flask or in a frozen state.

T25 Flask Coating Instructions:
*Coating T25 flasks:
1. Add 2 ml Universal Coating Solution into 3 T-25 flasks and ensure the entire interior surface is coated with the solution. After 30 minutes, dispose the Coating Solution by aspiration.
2. Gently rinse and aspirate the flask with Phosphate Buffer Solution 1XPBS. The flask is now ready for use (no need for overnight incubation when coated with Universal Coating Solution). Add fresh media to the flask; if the color changes from pink to yellow, discard the media and add fresh media to each flask.

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.