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GFP-Mouse Lung Fibroblasts

Size Price Quantity
1.0 Frozen Vial $790.00
Name of ProductsGFP-Mouse Lung Fibroblasts (GFP-MLFCs)
Catalog NumbercAP-m0023GFP
Product FormatFrozen Vial
Cell Number> 5x10[5] cells/vial
MLFCs were isolated from normal mouse lung tissue (3week old C57/BL6, male). GFP-MLFCs were selected from puromycin (5ug/ml) resistant MLFCs after being infected with lentiviruses expressing GFP. GFP-MLFCs are shipped in frozen vials (the cells are supplied @ passage 3). Fibroblast Growth Medium (FGM, contains 10% serum, cAP-44) is recommended for cell culture and these cells have a minimum average population doubling levels > 20 when cultured following the detailed protocol described below). C57/BL6 mice were obtained from Charles River (MA, USA) and are negative for known pathogens and mycoplasma
Cells are offered for Research Use Only.
Frozen Vials in a Dry Ice Package.
When you receive the dry ice package with cells in frozen vials, transfer the frozen vials of cells into a -80C freezer for short period storage or a liquid nitrogen tank for long- term storage.
A)Pre-coating of T25 flasks- Add 2ml each Quick Coating Solution (cAP-01) into a T25 flask to cover the whole surface of the flask, 5 mins later, dispose the excessive coating solution by aspiration and the flask is ready to be used (although solution containing other extracellular matrix, i.e. gelatin, collagen, and fibronectin, can be used, make sure to optimize the conditions in advance).
B)Thaw the frozen cell vial in a 37C water bath first, and then transfer the cells into one 15 ml falcon tube containing 10ml full medium, resuspend the cells, after being spinned down at 1000rpm for 8 mins, into one pre-coated T25 flask with 5ml of cAP-44 medium, cells usually become confluent within 1-2 days and ready to be passaged.
C)To passage the cells, rinse the cells in a T25 flask with 5ml HBSS (RT) twice; then add 2ml Trypsin/EDTA (RT) (cAP-23) into one T25 flask; gently dispose the excessive Trypsin/EDTA solution within 20 seconds by aspiration.
D)Leave the T25 flask with the cells at RT or 37C for 1 min (most cells usually will detach from the surface within 1-2 mins; or monitor the cells under a microscope until most of cells become rounded up, and then gently tap the flask against the bench surface, and the cells will move on the surface of the flask when monitoring under microscope.
E)Add 5ml Trypsin Neutralization Buffer and spin down the cells with 1000rpm for 8 mins.
G)Re-suspend the cell pellet with 10 or 15ml cAP-44 medium and transfer 5 ml each into 2 or 3 pre-coated T25 flasks (for 1/2 to 1/3 subculture ratio).
H)Change medium every 2 or 3days and the cells usually become confluent within 7 days (when split at a 1/3 ratio).
I)To prepare quiescent cells, when cells are nearly confluent, replace cAP-44 with Fibroblast Basal Medium (FBM, cAP-44B) containing 0.5%FBS for about 8-12hrs before your experiments.
Quick Coating SolutioncAP-01
Fibroblast Growth MediumcAP-44
Fibroblast Basal MediumcAP-44B
HBSS w/o Ca2+, Mg2+cAP-11
Cell Freezing Solution (FBS)cAP-22
Cell Freezing Solution (Non-FBS)cAP-22B
Trypsin/EDTA SolutioncAP-23
Trypsin Neutralization SolutioncAP-28
ITS (100x)cAP-26
L-Glutamine-MAXIMUM (100x) cAP-27
Human Plasma Fibronectin SolutioncAP-42
Handling human/aninal tissue derived products is potentially bio-hazardous, despite tested negative for HIV, HBV and HCV DNA; nonetheless, proper precautions must be taken to avoid inadvertent exposure.