For Research Use Only

CellBiologics

 Phone: (312) 226-8198			info@cellbiologics.com			http://www.cellbiologics.net 

Mouse Adenocarcinoma Epithelial Cells (Human Breast Cancer Origin)
Catalog No.  MBT31EP

Suggested Medium:  M6621 - Epithelial Cell Medium /w Kit – 500 ml

Product Description
Mouse Adenocarcinoma Epithelial Cells (Human Breast Cancer Origin) from Cell Biologics were isolated from human xenograft of pathogen-free laboratory nude mice. Human Breast adenocarcinoma epithelial cells (MDA-MB-231) were subcutaneously injected into the nude mice for 4-6 weeks and the tumor tissues were then harvested for cell culture. Mouse Adenocarcinoma Epithelial Cells (Human Breast Cancer Origin) were cultured in a T75 tissue culture flask with Cell Biologics’ Culture Complete Growth Medium for 3-5 days. Cells were detached from flasks and immediately cryo-preserved in vials. Cells will be delivered in a frozen vial and each vial contains 1x106 cells/1 ml.
Product Testing
Mouse Adenocarcinoma Epithelial Cells (Human Breast Cancer Origin) are characterized by immunofluorescent staining with antibodies of E-cadherin or ZO-1. Cells are negative for bacteria, yeast, fungi, and mycoplasma and can be expanded for 8-15 passages at a split ratio of 1:3 under the cell culture conditions specified by Cell Biologics. 
Laboratory Applications
Mouse Adenocarcinoma Epithelial Cells (Human Breast Cancer Origin) can be used in assays of standard biochemical procedures performed with cell cultures include cell-cell interaction, adhesion, PCR, Western blot, immunoprecipitation, 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.
Never can primary cells be kept at -20 °C.
Authorized Uses of Cell Biologics’ Products
Mouse Adenocarcinoma Epithelial Cells (Human Breast Cancer Origin) from Cell Biologics are distributed for 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
Although Mouse Adenocarcinoma Epithelial Cells (Human Breast Cancer Origin) are isolated from laboratory mice testing pathogen-free, investigators should handle the cells that they receive from Cell Biologics with caution and treat all animal cells as potential pathogens, since no test procedure can completely guarantee the absence of infectious agents.
Warranty and Liability
CELL BIOLOGICS’ guarantee applies only to your purchase of CELL BIOLOGICS' cells with CELL BIOLOGICS’ Media and Coating Solution for appropriate 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 10-30 min and then aspirate the excess solution before seeding cells.

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 200 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:

•	Remove and discard the cell culture media from the flask.
•	Flush the adherent layer 2 times using a 5 ml sterile pipette with sterile PBS (1X) without calcium and magnesium to dislodge loosely attached cells and remove fraction.
•	Remove and discard the wash solution from the flask.
•	Incubate cells with warm (37°C) 0.05% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6915) for 2-5 minutes. Use 3.0 ml of Trypsin-EDTA solution when collecting cells from T75 flasks, and 2 ml when using T25 flasks. 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 cells in fresh flasks or plates precoated with Gelatin-Based Coating Solution 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. Please note that 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 1-2 times whenever replacing the medium.

We recommend splitting primary cells at the follow ratio: 

•	The recommended split ratio for primary cells is 1:3.
•	A confluent monolayer of primary cells grown in a T75 flask may be expanded on a 6-well plate ready for use in experiments under the cell culture conditions specified by Cell Biologics.

Procedure for Freezing Cells

Materials:
•	1X Phosphate Buffered Saline (PBS-1X)
•	0.05% Trypsin-EDTA (1X) solution (Cell Biologics, Catalog No. 6915)
•	Tissue Culture Media
•	Cold Freezing Media (Cell Biologics, Catalog No. 6916).
•	Labeled Cryovials
•	Confluent cells 
------------------------------------------------------------------
•	Remove and discard the cell culture media from the flask. 
•	Flush the adherent layer with a 5 ml sterile pipette 2 times with sterile PBS (1X) without calcium and magnesium to dislodge loosely attached cells and remove fraction.
•	Remove and discard the wash solution from the flask.
•	Incubate cells with warm (37°C) 0.05% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6915) for 2-5 minutes. Use 3.0 ml of 0.25% Trypsin-EDTA solution when collecting cells from T75 flasks, and 2 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 10 ml of Cell Culture Medium supplemented with 5-10 % FBS to the flask (the FBS will neutralize the trypsin).
•	Centrifuge the cell suspension at 200 g for 5 minutes.
•	Remove supernatant with sterile Pasteur pipette.
•	Quickly re-suspend pellet by adding 1 ml freezing media per vial to be frozen.
•	Place vials in Nalgene "Mr. Frosty" freezing container containing100% isopropyl alcohol at -70-80 °C for 24 h.
•	Transfer vials to liquid N2 tank for indefinite storage.

We recommend freezing primary cells at the follow ratio: 

•	A confluent primary epithelial cells grown in a T75 flask may be frozen in 4-6 cryovials.
•	A confluent primary epithelial cells grown in a T25 flask may be frozen in 2-3 cryovial.