Immortalized Human Dermal Microvascular Endothelial Cells (SV40-HDMVECs)

We are thrilled to provide you with the following essential information that I'm sure you will find incredibly useful:

Product Name:
Immortalized Human Dermal Microvascular Endothelial Cells (SV40-HDMVECs)

Catalog Number:
UBPI003

Product Format:
Choose from either Frozen Vial or T-25 Flask

Cell Number:
Each vial contains >5x10^5 cells

Background:
HDMVECs cells were meticulously isolated from normal human skin tissues obtained from multiple donors. The SV40-HDMVECs were specially selected and engineered through infection with SV40 expressing lentiviruses, under the control of CMV, and made resistant to puromycin. Shipped at passage 2, the cells are stored in frozen vials. For optimal cell culture, we recommend Universal Endothelial Growth Medium (UEGM) containing 10% serum and growth supplements. These remarkable cells boast a minimum average population doubling level of > 40 when cultured following the detailed protocol provided below.

Cell Characterization:
1. 95%+ positive for Cytoplasmic VWF/Factor VIII by immunofluorescence
2. 95%+ positive for Cytoplasmic uptake of Di-I-Ac-LDL by immunofluorescence
3. 95%+ positive for Cytoplasmic PECAM1 by immunofluorescence
4. Tested negative for HIV-1, HBV, HCV, and mycoplasma

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

Shipping:
The cells are shipped in frozen vials in a dry ice package.

Handling Instructions:
Upon receiving the dry ice package with cells in frozen vials, please promptly transfer the vials to a -80°C freezer for short-term storage or a liquid nitrogen tank for long-term storage.

We are confident that these superb SV40-HDMVECs will meet and exceed your expectations, contributing significantly to your research endeavors.

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.