For Research Use Only

Human Breast Tumor-Associated Endothelial Cells
Catalog No.  HC-6020

Suggested Medium: Complete Human Endothelial Cell Medium– 500ML
Catalog No.  H1168
	
Product Description
Human Breast Tumor-Associated Endothelial Cells from Cell Biologics are isolated from human breast tumor tissue. Cells are grown in T25 tissue culture flasks pre-coated with gelatin-based solution for 2 min and incubated in Cell Biologics’ Culture Complete Growth Medium generally for 3-7 days. Cultures are then expanded. Cells at passage 3 are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 0.5x106 cells per ml and is delivered frozen. The method we use to isolate primary endothelial cells was developed based on a combination of established and our proprietary methods. These cells are pre-coated with PECAM-1 antibody, following the application of magnetic beads pre-coated with secondary antibody. 
Product Testing
Human Breast Tumor-Associated Endothelial Cells from Cell Biologics display typical cobblestone with large dark nuclei appearance under light microscopy. Cells are tested for expression of endothelial cell marker using antibody, CD31 (Catalog No. 550389, BD; CD31/PECAM-1 PE-conjugated Antibody, Catalog No. FAB3567P, R&D) or VE-Cadherin (FITC-VE-cadherin Catalog No. 560411, BD) by immunofluorescence staining or FACS. All cells test negative for mycoplasma, bacteria, yeast, and fungi. HIV-1, hepatitis B and hepatitis C are not detected for all donors and/or cell lots. Per request, a Certificate of Analysis will be provided for each cell lot purchased. Cells can be expanded for 2-4 passages under the cell culture conditions specified by Cell Biologics. Repeated freezing and thawing of cells is not recommended.
Laboratory Applications
Human Breast Tumor-Associated Endothelial Cells can be used in assays of cell to cell adhesion, migration, vascular tube formation. Standard biochemical procedures performed with endothelial cell cultures include RT-PCR, Western blotting, immunoprecipitation, immunofluorescent staining, immunofluorescent flow cytometry, or generating cell derivatives for desired research applications.
Storage of Cell Biologics’ Products
Cell Biologics ships frozen cells on dry ice. On receipt, immediately transfer frozen cells to liquid nitrogen
(-180 °C) until ready for experimental use. Live cell shipment is also available on request. Primary cells should never be kept at -20 °C freezer.
Authorized Uses of Cell Biologics’ Products
Human Breast Tumor-Associated Endothelial Cells from Cell Biologics are distributed for internal research purposes only. Our products are not authorized for human use, for in vitro diagnostic procedures or for therapeutic procedures. Transfer or resale of any Cell Biologics’ cells or products from the purchaser to other markets, organizations or individuals is prohibited by Cell Biologics, without the company’s written consent. 
Cell Biologics’ Terms and Conditions must be accepted before submitting an order.
Disclaimer
Appropriate safety procedures should always be used with this material. Although Human Breast Tumor-Associated Endothelial Cells are isolated from normal human tissue, investigators should handle the cells that they receive from Cell Biologics with caution and treat all primary cells as potential pathogens, since no test procedure can completely guarantee the absence of infectious agents. The entire text of discussing Biosafety in Microbiological and Biomedical Laboratories, 5th ed. is available online at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.
Warranty and Liability
Cell Biologics’ guarantee applies only to your purchase of Cell Biologics’ human endothelial cells with Cell Biologics’ Media and Coating Solution, for appropriate endothelial cell culture and cell testing following Cell Biologics’ online protocols within 35 days from the date of product delivery.

Primary Cell Culture Protocol

All cell culture procedures must be conducted in a bio-safety cabinet.
Any and all media, supplements, and reagents must be sterilized by filtration through a 0.2 µm filter. 
Use aseptic technique to prevent microbial contamination.
Cryo-preserved cells must be stored in liquid nitrogen or seeded immediately upon arrival.

Medium
Review the information provided on the Cell Biologics website about appropriate culture media (e.g. serum and other supplements). Use pre-warmed (37°C) cell culture media (30-50 ML) to recover cryo-preserved cells and when changing media or splitting cells.

Coating of flasks or dishes
Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950) for 2 min and then aspirate the excess solution before seeding cells.

Handling of Arriving Live Cells
When you receive the live cells in a T25 or T75 flask, remove the sticker from the filter cap, and keep the flask with 6-20 ml existing medium in 37°C CO2 incubator for 1 hour before replacing the desired Cell Biologics' cell culture medium. Either split the 95-100% confluent cells from a T25 flask to 2 of T25 flasks after 1 hour or let the cells grow in the T25 flask with the desired Medium (such as H1168) for 12 hours before subculturing cells. The recommended split ratio for primary cells is 1:2.

Cell recovery from cryovial
•	Quickly thaw cells in cryo-vial by incubating them in a 37°C water bath for <1 min until there is just a small bit of ice left in the vial.
•	Promptly remove the vial and wipe it down with 70% ethanol.
•	Transfer cells from the vial to a sterile centrifuge tube. Add 8-10 ml of pre-warmed Cell Biologics Cell Culture Medium. Flush the vial with an additional 0.5-1 ml of medium to ensure complete transfer of cells to the centrifuge tube.
•	Centrifuge cells at 150 g for 5 minutes.
•	Aspirate the supernatant and resuspend the cell pellet in 6 ml of Cell Biologics’ Cell Culture Growth Medium.
•	Add resuspended cells into a T25 flask pre-coated with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950).
•	Place the T25 flask in a humidified, 5%-CO2 incubator at 37°C. 
•	Change culture media the following day to remove non-adherent cells and replenish nutrients.
•	Change cell culture medium every day when cells are >70% confluent.
•	Cells should be checked daily under a microscope to verify appropriate cell morphology.

Expansion of cultured primary cells
•	Flush the adherent layer with a 5 ml sterile pipette 1-2 times to dislodge loosely attached cells.
•	Remove and discard the cell culture media from the flask.
•	Wash adherent cells 2 times with 10 ml of sterile PBS (1X) without calcium and magnesium to remove nonadherent cells or fraction.
•	Remove and discard the wash solution from the flask.
•	Incubate cells with warm (37°C) 0.05% Trypsin-EDTA (1X) solution (Cell Biologics, Catalog No. 6915) for 1 minute. Use 2.0 ml of 0.05% Trypsin-EDTA solution when collecting cells from a T75 flask, and 1.0 ml when using a T25 flask. As soon as cells have detached (the flask may require a few firm gentle taps), add 8-10 ml of Cell Biologics Cell Culture Medium supplemented with 5-10 % FBS to a T25 or T75 flask (the FBS will neutralize the trypsin).
•	Plate flasks or plates in a humidified, 5%-CO2 incubator at 37°C. 
•	Change culture media the following day to remove non-adherent cells and replenish nutrients.
•	Cells should be checked daily under a microscopy to verify appropriate cell morphology.
•	Change culture medium every 24-48 hours. 
•	The medium should be changed every day when cells are >70% confluent to remove non-adherent cells and replenish nutrients.
•	Pre-wash cells with 1X PBS 2 times whenever replacing the medium.

We recommend splitting primary cells at the follow ratio
•	The recommended split ratio for primary cells is 1:2.
•	A confluent monolayer of primary cells grown in a T75 flask may be expanded on one 6-well plate ready for use in experiments under the cell culture conditions specified by Cell Biologics.

Note: 
•	You may let cells over-growing for 24-48 h after cells reach confluence before doing any cell testing, cell staining, FACS, or designed experiments.
•	Cells can be filtered through a 40 micron cell strainer (BD 352340) just before passaging cells or doing experiments to remove any clumps of cells and most of the dead/floating large cells.
•	Please send us the cell images (>90% confluence) if you have any question or problem with cultured cells.