Primary Human Brain Microvascular Endothelial Cells

Introducing our exceptional product, the Human Brain Microvascular Endothelial Cells, catalog number UBP02. These cells are of the utmost quality, isolated from normal Human Brain Tissue, with passage 1 cells shipped in a proliferating culture with a confluence of over 90% in a T25 flask or in a frozen vial.

The cells come with an impressive guarantee of greater than 95% positivity for cytoplasmic VWF/factor VIII, Di-I-Ac-LDL, and PECAM1 by immunofluorescence, making them the ideal choice for your research needs.

It's essential to note that all cell strains have been meticulously tested and confirmed negative for HIV, HBV, HCV, and mycoplasma. Furthermore, proper precautions should be taken during handling, as working with human-derived products could pose biological hazards. We strongly recommend wearing appropriate protective equipment such as gloves and safety glasses and following universal procedures for handling products of human origin to minimize the risk of contamination.

To ensure successful culture, we highly recommend the use of our Universal ENDO-Growth medium, which contains 10% serum and growth supplements, and has been shown to yield exceptional results with an average population doubling level of greater than 15 when cultured. 

Upon receipt of the cells, it is crucial to follow the provided instructions and leave the flask in a 37°C CO2 incubator for 1 hour, subsequently replacing the transport medium with fresh Universal ENDO-Growth medium and allowing the cells to grow for 24 hours before subculture.

We take pride in offering cells that can be confidently used for research purposes. As such, we emphasize that the Human Brain Microvascular Endothelial Cells are designated for research use only. The cells are shipped in a proliferating culture in T25 flasks or a frozen Vial, complete with detailed instructions for flask coating and cell subculture procedures to ensure seamless and successful handling.

In addition to the product details, we understand the importance of clarity and guidance in using our products effectively. The provided T25 flask coating and cell subculture procedures offer clear steps and recommendations to ensure optimal handling and maintenance of the cells, facilitating a smooth and efficient research process.

We are thrilled to offer these exceptional Human Brain Microvascular Endothelial Cells for your research endeavors. With their impressive qualities and comprehensive instructions, we are confident that these cells will be a valuable asset to your laboratory and contribute to the advancement of your research.

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.