B129 Mouse Lung Mesenchymal Stem Cells

Catalog No. B129-7211

Suggested Medium: Cell Culture Medium /w Kit (500 ml)

Catalog No. M5567

Product Description

Mouse Lung Mesenchymal Stem Cells are derived from the lung tissues of pathogen-free laboratory adult mice. Mouse Lung Mesenchymal Stem Cells are grown in T25 tissue culture flasks pre-coated with gelatin-based solution for 0.5 hour and incubated in Cell Biologics’ Cell Culture Medium for 7-15 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. Mouse Lung Mesenchymal Stem Cells are negative for bacteria, yeast, fungi, and mycoplasma. Cells can be expanded 3-8 passages by the ratio of 1:3 under the cell culture conditions specified by Cell Biologics. Repeated freezing and thawing of cells is not recommended.

Mouse Lung Mesenchymal Stem Cells are tested for expression of markers using antibodies, CD44, Sca-1 and CD29 positive by flow cytometry.

Laboratory Applications

Standard biochemical procedures performed with cell cultures include RT-PCR, Western blotting, immunoprecipitation, immunofluorescent staining, flow cytometry or generating cell derivatives for desired research applications.

Storage of Cell Biologics Products 

Cell Biologics will ship 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 Lung Mesenchymal Stem 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 Biologics without the company’s written consent.  Cell Biologics’ Terms and Conditions must be accepted before submitting an order.

Disclaimer

Although Mouse Lung Mesenchymal Stem Cells 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.

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 medium 4-6 hours after thawing cells to ensure >90% cells are attached.
• Also change culture media the following day to remove non-adherent cells and replenish nutrients.
• Change cell culture medium everyday when cells are >60% confluent.
• Cells should be checked daily under a microscope to verify appropriate cell morphology.

Expansion of cultured mouse primary cells:

• Flush the adherent layer with a 5 ml sterile pipette 3 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) 0.25% Trypsin-EDTA (1X) solution for 1 minutes. Use 2.0 ml of 0.25% Trypsin-EDTA solution when collecting cells from T75 flasks, and 1.0 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 medium 4-6 hours after seeding cells to ensure >90% cells are attached. Also 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.
• Medium should be changed every day when cells are >60% 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 murine 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 (Invitrogen catalog number 25300)
• Tissue Culture Media
• Cold Freezing Media (10% dimethyl sulfoxide (DMSO), 20% FBS and 70% culture medium)
• Labeled Cryovials
• Confluent cells

          --------------------------------------

• Flush the adherent cell 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 (5X) solution for 1 minutes. Use 2.0-3.0 ml of 0.25% Trypsin-EDTA solution when collecting cells from a T75 flask, and 1.0-1.5 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.
• 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 cells grown in a T75 flask may be frozen into 3 cryovials.

• A confluent primary cells grown in a T25 flask may be frozen into 2 cryovial.