MEF Feeder Cells (Puromycin-resistant) Cell Line
MEF Feeder Cells (Puromycin-resistant)
Synonyms
MEF Feeder Cells (Puromycin-resistant); MEF Feeder Cells (Puromycin-resistant) cell line
Concentration
1 mL, 5 x 10^6 cells/mL in 90% FBS, 10% DMSO (varies by lot)
Application Notes
Puromycin-resistant Embryonic Fibroblast (MEF) feeder cells are used for the maintenance of stable Puromycin-resistant mouse or human ES cells in the undifferentiated state. The cells must be mitotically inactivated prior to the addition of ES cells, such as treatment with mitomycin C (2-4 hr, 10 ug/mL).
Methods
I. Establishing MEF Feeder Cell Cultures from Frozen Cells
1. Place 10 ml of complete DMEM growth medium in a 50-ml conical tube. Thaw the frozen cryovial of cells within 1-2 minutes by gentle agitation in a 37 degree C water bath. Decontaminate the cryovial by wiping the surface of the vial with 70% (v/v) ethanol.
2. Transfer the thawed cell suspension to the conical tube containing 10 mL of growth medium.
3. Collect the cells by centrifugation at 1000 rpm for 5 minutes at room temperature. Remove the growth medium by aspiration.
4. Resuspend the cells in the conical tube in 15 mL of fresh growth medium by gently pipetting up and down.
5. Transfer the 15 mL of cell suspension to a T-75 tissue culture flask. Place the cells in a 37 degree C incubator at 5% CO2.
6. Monitor cell density daily. Cells should be passaged when the culture reaches 95% confluence.
II. Freezing MEF Feeder Cells
1. Trypsinize cells and resuspend cell pellet in cold Freeze Medium at twice the desired final cell concentration.
2. Aliquot 1 mL of cells into sterile cryovials and place cryovials immediately into freezing container. Store overnight at -80 degree C.
3. Transfer frozen vials to -135 degree C freezer or liquid nitrogen.
III. Mitomycin C Treatment and Preparation of Feeder
1. Culture cells to 90% confluence. Wash it once with sterile PBS.
2. Add 10 ug/mL Mitomycin C (Sigma), incubate for 2 hrs.
3. Wash 3 times with sterile PBS to remove Mitomycin.
4. After dissociation by Trypsin, the Mitomycin-treated MEFs can be freezed and stored in liquid nitrogen, or used as feeder by plating them at 75 000 cells/cm2 in gelatin-coated tissue culture dishes for one day.
1. Place 10 ml of complete DMEM growth medium in a 50-ml conical tube. Thaw the frozen cryovial of cells within 1-2 minutes by gentle agitation in a 37 degree C water bath. Decontaminate the cryovial by wiping the surface of the vial with 70% (v/v) ethanol.
2. Transfer the thawed cell suspension to the conical tube containing 10 mL of growth medium.
3. Collect the cells by centrifugation at 1000 rpm for 5 minutes at room temperature. Remove the growth medium by aspiration.
4. Resuspend the cells in the conical tube in 15 mL of fresh growth medium by gently pipetting up and down.
5. Transfer the 15 mL of cell suspension to a T-75 tissue culture flask. Place the cells in a 37 degree C incubator at 5% CO2.
6. Monitor cell density daily. Cells should be passaged when the culture reaches 95% confluence.
II. Freezing MEF Feeder Cells
1. Trypsinize cells and resuspend cell pellet in cold Freeze Medium at twice the desired final cell concentration.
2. Aliquot 1 mL of cells into sterile cryovials and place cryovials immediately into freezing container. Store overnight at -80 degree C.
3. Transfer frozen vials to -135 degree C freezer or liquid nitrogen.
III. Mitomycin C Treatment and Preparation of Feeder
1. Culture cells to 90% confluence. Wash it once with sterile PBS.
2. Add 10 ug/mL Mitomycin C (Sigma), incubate for 2 hrs.
3. Wash 3 times with sterile PBS to remove Mitomycin.
4. After dissociation by Trypsin, the Mitomycin-treated MEFs can be freezed and stored in liquid nitrogen, or used as feeder by plating them at 75 000 cells/cm2 in gelatin-coated tissue culture dishes for one day.
Medium
1. Culture Medium: D-MEM (high glucose), 10% fetal bovine serum (FBS), 2 ug/mL Puromycin, 0.1 mM MEM Non-Essential Amino Acids (NEAA), 2 mM L-glutamine, 1% Pen-Strep (optional)
2. Freeze Medium: 90% FBS, 10% DMSO
2. Freeze Medium: 90% FBS, 10% DMSO
Shipping Note
Product available only in the USA.
Quality Control
Puromycin-resistant Embryonic Fibroblast (MEF) feeder cells are cultured in medium containing 2 ug/mL Puromycin. This cryovial contains at least 5.0 × 10^6 MEF feeder cells as determined by morphology, trypan-blue dye exclusion, and viable cell count. The MEF feeder cells are tested free of microbial contamination.
Dry Ice Shipment
Extra charge fee may add to your shipping cost as dry ice is required to ship this product.
Preparation and Storage
Liquid nitrogen
Note: For best results begin culture of cells immediately upon receipt. If this is not possible, store at -80 degree C until first culture. Store subsequent cultured cells long term in liquid nitrogen.
Note: For best results begin culture of cells immediately upon receipt. If this is not possible, store at -80 degree C until first culture. Store subsequent cultured cells long term in liquid nitrogen.
Related Product Information for MEF Feeder Cells (Puromycin-resistant) cell line
• Murine Embryonic Fibroblasts allow stem cell culture without LIF
• Presented as 5 x 10^6 cells in 1 mL
• Must be mitotically inactivated prior to addition of ES cells
Embryonic stem (ES) cells have been derived from the inner cellmasses (ICM) of blastocysts in many species. They are capable of unlimited, undifferentiated proliferation on feeder cell layers and remain karyotypically normal and phenotypically stable. In addition, ES cells have the ability to differentiate into a wide variety of cell types in vitro and in vivo. In mES cell culture, the feeder layer can be replaced by the addition of LIF in the growth medium. However, LIF does not have the same effect on hES cell culture as mES. Therefore, both the derivation and maintenance of hES cells require the use of feeder cells.
• Presented as 5 x 10^6 cells in 1 mL
• Must be mitotically inactivated prior to addition of ES cells
Embryonic stem (ES) cells have been derived from the inner cellmasses (ICM) of blastocysts in many species. They are capable of unlimited, undifferentiated proliferation on feeder cell layers and remain karyotypically normal and phenotypically stable. In addition, ES cells have the ability to differentiate into a wide variety of cell types in vitro and in vivo. In mES cell culture, the feeder layer can be replaced by the addition of LIF in the growth medium. However, LIF does not have the same effect on hES cell culture as mES. Therefore, both the derivation and maintenance of hES cells require the use of feeder cells.
Product Categories/Family for MEF Feeder Cells (Puromycin-resistant) cell line
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Product Notes
The MEF Feeder Cells (Puromycin-resistant) (Catalog #AAA168047) is a Cell Line and is intended for research purposes only. The product is available for immediate purchase. Puromycin-resistant Embryonic Fibroblast (MEF) feeder cells are used for the maintenance of stable Puromycin-resistant mouse or human ES cells in the undifferentiated state. The cells must be mitotically inactivated prior to the addition of ES cells, such as treatment with mitomycin C (2-4 hr, 10 ug/mL). Researchers should empirically determine the suitability of the MEF Feeder Cells (Puromycin-resistant) for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "MEF Feeder Cells (Puromycin-resistant), Cell Line" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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