Primary Human Cord Blood CD34+ Endothelial Cells

- Product Name: Human CD34 positive Cord Blood cells Derived Endothelial Cells (HCD34+CBDECs)
- Catalog Number: UBP23
- Product Format: Available in Frozen Vial or T-25 Flask
- Cell Number: Each vial contains over 5x10^5 cells

Description:
Our HCD34+CBDECs are a product of utmost quality, derived from CD34 positive human cord blood cells and cultured under endothelial cell inducing medium. When you receive them, the cells are carefully preserved in frozen vials at passage 1 or T-25 Flask. To nurture them, we recommend using our Universal Endothelial Growth Medium (UEGM) enriched with 10% serum and growth supplements. Our cells boast a minimum average population doubling level of over 16 when cultured following the provided protocol.

Characterization of the Cells:
1. Our cells display over 95% positivity for Cytoplasmic VWF/Factor VIII by immunofluorescence
2. They also exhibit over 95% positivity for Cytoplasmic uptake of Di-I-Ac-LDL by immunofluorescence
3. Additionally, they showcase over 95% positivity for Cytoplasmic PECAM1 by immunofluorescence
4. Our HCD34+CBDECs are meticulously tested and confirmed to be negative for HIV-1, HBV, HCV, and mycoplasma

Product Usage:
These exceptional cells are offered for Research Use Only.

Shipping:
Rest assured that the cells are shipped in frozen vials within a dry ice package, ensuring their preservation during transit.

Handling of Arriving Cells:
Upon receiving the dry ice package housing the frozen vials, we recommend promptly transferring the vials into a -80°C freezer for short-term storage or a liquid nitrogen tank for long-term storage.

Cell culture instructions for a T-25 flask:

1. Ensure that the flask is at 90% confluence. If not, remove the transport medium and add 5 ml of fresh media to the flask. Place the flask in a 37°C incubator until the cells reach 90% confluence, changing the media every 2 days.

2. If the flask is at 90% confluence, remove the transport media from the flask.

3. Rinse the T-25 flask containing cells with 5 ml 1X PBS.

4. Gently aspirate out the 1X PBS after rinsing and discarding.

5. Add 2 ml of room temperature Universal Detachment solution to the T-25 flask containing cells, ensuring that 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 15 ml of Universal ENDO-Growth Medium and transfer them 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 when Universal Cell Detachment solution is used.

9. For proliferating cell culture, change the Universal ENDO-Growth medium 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.