Human Dermal Lympathic Microvascular Endothelial Cells

 Human Dermal Lymphatic Microvascular Endothelial Cells, available under the catalog number UBP03. These cells are stored in liquid nitrogen in a frozen vial format and have an impressive cell confluence of over 90%.

When dealing with human-derived products, it is crucial to be aware of potential biological hazards. Be assured that all cell strains have undergone rigorous testing and have tested negative for HIV, HBV, and HCV DNA in diagnostic tests. Emphasizing the importance of taking proper precautions, we strongly encourage the use of appropriate protective equipment, such as gloves and safety glasses, to minimize any risk of exposure to ensure safety and peace of mind. Adhering to universal procedures for handling human-origin products is imperative as a fundamental measure against contamination.

In terms of general information, these exceptional cells have been isolated from normal Human Dermal Lymphatic Microvascular Endothelial tissue. Shipped in proliferating culture, our Passage 1 cells boast an incredible confluence of over 90%. For optimal cultivation, we recommend using the Universal ENDO-Growth medium containing 5% serum and growth supplement. Cultured cells exhibit an average population doubling level exceeding 14.

Upon receipt of the cells, it is advisable to place the flask in a 37°C CO2 incubator for 1 hour. Following this, replace the transport medium with fresh our Universal ENDO-Growth medium and allow the cells to grow for 24 hours before subculture.

The remarkable cellular characteristics include a greater than 95% positive immunofluorescence for Cytoplasmic VWF/factor VIII, Cytoplasmic uptake of DiI-Ac-LDL, and Cytoplasmic PECAM1. Furthermore, these cells have been tested negative for HIV-1, HBV, HCV, and mycoplasma.

It's important to note that Human Dermal Lymphatic Microvascular Endothelial Cells are designated for research use only and are shipped in a frozen vial format or in T-25 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.