Cell Type, Endothelial Cells et al.
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Product Description

C57BL/6 Mouse Aortic Endothelial Cells from Cell Biologics are isolated from tissue of pathogen-free laboratory mice. C57BL/6 mouse aortic endothelial cells are grown in T75 tissue culture flasks pre-coated with 0.2% gelatin for 0.5 hour and incubated in Cell Biologics Culture Complete Growth Medium generally for 3-7 days. Cultures are then expanded.  Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials.  Each vial contains at least 1x106 cells per ml and is delivered frozen. 

 

Product Testing

We test each batch of C57BL/6 mouse Aortic Endothelial Cells for expression of markers using the antibodies, Ve-cadherin or vwf by immunofluorescence staining or flow cytometry.  We also test for uptake of Dil-Ac-LDL, a functional marker for endothelial cells. C57BL/6J mouse Aortic Endothelial Cells are negative for bacteria, yeast, fungi, and mycoplasma, and are guaranteed to expand by at least 3-6 passages under the cell culture conditions specified by Cell Biologics.

 

Laboratory Applications

C57BL/6 Mouse Aortic Endothelial Cells can be used in assays of cell-cell adhesion, migration, vascular endothelial tube formation: an in vitro matrigel angiogenesis assay or agiogenesis in vivo within a Matrigel plug in mice.  Standard biochemical procedures performed with endothelial cell cultures include RT-PCR, Western blotting, immunoprecipitation, immunofluorescent staining or flow cytometry.

 

Storage of Cell Biologics Products

Cell Biologics ships frozen cells on dry ice.  On receipt, immediately transfer frozen cells to liquid nitrogen until ready for experimental use.  Live-cell shipment is also available on request.

 

Authorized Uses of Cell Biologics Products

C57BL/6 mouse aortic endothelial cells 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 BiologicsCell Biologics’ Terms and Conditions must be accepted before submitting an order.

 

Disclaimer

Although C57BL/6 mouse aortic endothelial cells are isolated from laboratory mice testing pathogen-free, investigators should handle the cells that they receive from Cell Biologics with caution, since no test procedure can completely guarantee the absence of infectious agents.

 



General protocol for the culture of mouse primary cells

 

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 to recover cryo-preserved cells or when changing media or splitting cells.

 

Coating of flasks or dishes:

 

Coat sterile culture dishes or flasks with 0.2% gelatin (Sigma catalog No. G1393).  Prepare the gelatin solution in pre-warmed (37°C), sterile PBS (1X) containing no calcium and magnesium.  Apply gelatin solution for 10-30 min and then aspirate the excess solution before seeding cells.

 

 

Protocol for Recovery of Frozen Cultured Cells


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 10 ml of pre-warmed 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 5-7 ml of Cell Culture Growth Medium.

 

Add resuspended cells into a T25 flask pre-coated with 0.2% gelatin.

 

Place the T25 flask in a humidified, 5%-CO2 incubator at 37°C. Change medium on the next day.

 

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 3-5 times to dislodge loosely attached cells.

 

Remove and discard the cell culture media from the flask.

 

Wash adherent cells 2-3 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 (Invitrogen catalog number 25300) for 2-3 minutes. Use 3.0 ml of 0.05% Trypsin-EDTA solution when collecting cells from a T75 flask, and 2.0 ml when using a T25 flask. 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.

 

Plate cells in a fresh flask or on a multiwell culture plate pre-coated with 0.2% gelatin and add 12-15 ml of the Cell Culture Medium.

 

Cells should be checked daily under a microscopy to verify appropriate cell morphology.

 

Change culture medium every 24-48 hours. Pre-wash cells with 1X PBS whenever replacing the medium.

 

 

We recommend splitting cells at the follow ratio:

 

The recommended split ratio for primary murine cells is 1:2.

 

A confluent monolayer of primary cells grown in a T75 flask may be expanded on a multiwell culture plate ready for use in experiments under the cell culture conditions.

 

One confluent monolayer of primary cells grown in a T25 flask may be split into two T25 flasks or into one T75 flask.

One confluent monolayer of primary cells grown in a T75 flask may be split into two T75 flasks, or into two-three 100 mm2 culture dishes, or into four-five 60 mm2 culture dishes.

 

 

Cell Freezing procedure:

 

Materials:

 

·         Phosphate Buffered Saline (PBS)

·         0.05% Trypsin-EDTA (1X) solution (Invitrogen catalog number 25300)

·         Tissue Culture Media

·         Cold Freezing Media (10% dimethylsulfoxide, DMSO and 20% FBS, and 70% culture medium)

·         Labeled Cryovials (~3 per 100-mm plate for)

·         T25 flask or T75flask confluent cells

 

Flush the adherent layer with a 5 ml sterile pipette 3-5 times to dislodge loosely attached cells.

 

Remove and discard the cell culture media from the flask.

 

Wash adherent cells 2-3 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) Trypsin-EDTA (1X) solution for 1-3 minutes. Use 2.0-3.0 ml of 0.05% Trypsin-EDTA solution when collecting cells from T75 flasks, and 1.5-2.0 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.