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Immortalized Mouse EGFR Mutant M3-/- Epidermal Keratinocytes

SKU : BHC10900729

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Applied Biological Materials (abm) Inc.

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Mfr.No.	T0461
Description	The Immortalized Immortalized Mouse EGFR Mutant M3-/-?pidermal Keratinocytes were derived from primary keratinocytes of newborn EGFR null mice. The primary keratinocytes were immortalized in subsequent cultures after passaging for an extended period. The immortalized cells display similar phenotype to the primary keratinocytes including the cobblestone-shape morphology.?br />The Immortalized Mouse EGFR Mutant M3-/- Epidermal Keratinocytes are non-tumorigenic. They also differentiate with increased calcium, maintain a relatively stable karyotype no more aberrant than cultured primary keratinocytes, and produce epidermis upon orthotopic grafting onto nude mouse hosts. The M3 muscarinic acetylcholine receptor is knocked out in these cells. Upon introduction of v-rasHa oncogene, the cells produce squamous cell carcinoma in orthotopic skin grafts. These cells are useful in studies focusing on epidermal keratinocyte biology and pathology.
Cell Type	Immortalized Cells
Biosafety Level	2
Depositor	Creighton University
Disclaimer	1. All test parameters provided in the CoA are conducted using abm's standardized culture system and The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 ?g, Cat.# C207, $450.00) or cell lysate (100 ?g, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final. 2. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of order placement. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. 3. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic or any other non-RUO application(s). 4. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature are provided for informational purposes only. abm does not warrant that such information has been shown to be accurate. 5. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period".
Donor History	EGFR null mice, newborn
Expression Region	Keratin 14, Keratin 6
Growth Properties	Adherent, cobblestone
Growth Conditions	Use of PriCoat?T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Thaw the cells with PriGrow XI (TM011) + 8% FBS + 60 ? CaCl? (added fresh each time) + 1% Penicillin/Streptomycin Solution (G255), 37.0?, 8% CO? for optimal attachment. After 24h, change media to PriGrow XI (TM011) + 8% chelexed FBS + 1.4% FBS + 5 ng/ml rhFGF7 (Z100595) (freshly added) + 1% Penicillin/Streptomycin Solution (G255), 37.0?, 8% CO? Steps to thaw the cells: 1. Thaw the cells in RT water until only a few ice crystals are left 2. Put the cells in 5 ml of RT PriGrow XI (TM011) + 8% FBS 3. Centrifuge the cells 1500 rpm for 5 min (need this step to remove inhibitors for attachment) 4. Resuspend the pellet in RT Prigrow XI (TM011) + 8% FBS + 1% P/S with freshly added 60 ? CaCl? and put the cells in a culture vessel at 37.0?, 8% CO? 5. After 18h - 24h, change to PriGrow XI (TM011) + 8% chelexed FBS + 1.4% FBS + 5 ng/ml rhFGF7 (Z100595) (freshly added) + 1% Penicillin/Streptomycin Solution (G255), 37.0?, 8% CO? for growth of the cells.
Quantity	1x106 cells / 1.0 ml
Tissue	Skin
Morphology	Polygonal
Notes	1. Visually examine the packaging containers for signs of leakage or breakage. 2. Immediately transfer frozen cells from dry ice packaging to a temperature below -130?, preferably in liquid nitrogen vapor phase storage, until ready for use.To ensure the highest level of viability, thaw the vial and initiate culture as soon as possible upon receipt. If continued storage is desired, the vial should only be stored below -130? or in liquid nitrogen vapor phase. Do not store at -70?, as it will result in loss of viability.
Organism	Mouse (M. musculus)
Population Doubling	40 - 50 hours
Species	Human (H. sapiens)
Propagation	Use of PriCoatTM T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels unless otherwise specified in the Propagation Requirements below.The base medium for this cell line is Prigrow I medium available at abm, Cat. No. TM001. To make the complete growth medium, add the following components to the base medium: fetal bovine serum (TM999)* to a final concentration of 10%, Penicillin/Streptomycin Solution (G255) to a final concentration of 1%, endothelial cell growth supplement (ECGS) to a final concentration of 12.5 µg/ml, heparin to a final concentration of 90 µg/ml, and L-Glutamine Solution (200 mM) (G275) to a final concentration of 2 mM.Change media every 2-3 days.Carbon dioxide (CO2): 5%, Temperature: 37.0°C.* Do not use heat-inactivated FBS for cell culture unless specified otherwise.
Cryopreservation	Cryopreservation Medium (TM024), or complete growth media with 10% DMSO.
Quality Control	1) Real Time PCR

Applications Range	Research Use Only.

Shipping	Ship with dry ice.
Product Format	Frozen
Product Use	This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Storage	Vapor phase of liquid nitrogen, or below -130?.

Thawing Protocol	1. Thaw cells quickly in a 37? water bath while agitating gently (maximum 2 minutes). The vial cap should be kept above the water level to minimize the risk of contamination. 2. Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. From this point onwards, all operations should be strictly carried out inside a biological safety cabinet using aseptic conditions. 3. Transfer the cell suspension into a 15ml sterile conical tube containing 5ml of pre-warmed, complete growth media. Centrifuge cells at 125xg for 5-7 minutes. 4. Aspirate the supernatant without disturbing the cell pellet. Re-suspend the cell pellet in the recommended pre-warmed, complete growth media and dispense into a T25 culture flask. 5. Incubate the cells at the recommended conditions.
Subculture Protocol	Volumes given below are for a T75 flask; proportionally increase or decrease the volume as required per culture vessel size. Subculture cells once the culture vessel is 80% confluent. 1. Aspirate the culture media, and add 2-3ml of pre-warmed 0.25% Trypsin-EDTA to the culture vessel. 2. Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes). Cells that are difficult to detach can be put in 37?, for several minutes to facilitate detachment. 3. Neutralize Trypsin-EDTA by adding an equal volume of the complete growth media into the culture vessel. 4. Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 125xg for 5 minutes. The actual centrifuge duration and speed may vary depending on the cell type. 5. Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new culture vessels, as desired. 6. Incubate the cells at the recommended conditions.

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SKU	Supplier No.	Size	Your price	Quantity
BHC10900729	T0461	1x10^6 cells / 1.0 ml	-		Add to Quote

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