Human Brain Microvascular Endothelial Cell GFP-AEQ in Mitochondria

 Human Brain Microvascular Endothelial Cells GFP-AEQ in Mitochondria (HBMVEC-GFP-AEQ)
- Catalogue Number: UBP02AEQGFP
- Product Format: Frozen Vial OR T-25 Flask
- Cell Number: Each vial contains over 5x10[5] cells

Let's delve into the details! These exceptional HBMVEC-GFP-AEQ cells were isolated from the normal human cerebral cortex and carefully selected by puromycin from HBMVECs after being infected with special lentiviruses that express GFP-AEQUORIN in mitochondria. The cells, provided at passage 2, are shipped in frozen vials and boast a remarkable minimum population doubling level of >15 when following the provided detailed protocol.

Characterization of the Cells:
1. Over 95% positive for cytoplasmic VWF/Factor VIII by immunofluorescence
2. Over 95% positive for cytoplasmic uptake of Di-I-Ac-LDL by immunofluorescence
3. Over 95% positive for cytoplasmic PECAM1 by immunofluorescence
4. HBMVECs are negative for HIV-1, HBV, HCV, and mycoplasma

For optimal cell culture, we recommend using our Universal Endothelial Growth Medium containing 10% serum and growth supplements.

Shipping:
Your cells will be shipped in frozen vials carefully packaged in dry ice.

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

Remember, these cells are offered for Research Use Only, opening the doors for exciting discoveries and advancements! 🧪🔬

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.