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Complete Growth Medium
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The base medium for this cell line is ATCC Hybri-Care Medium, Catalog No. 46-X. Hybri-Care Medium is supplied as a powder and should be reconstituted in 1 L cell-culture-grade water and supplemented with 1.5 g/L sodium bicarbonate. To make the complete growth medium, add the following components to the base medium: 30 ng/ml mouse EGF; fetal bovine serum to a final concentration of 10%.
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Subculturing
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Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
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Remove and discard culture medium.
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Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
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Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
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Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
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Add appropriate aliquots of the cell suspension to new culture vessels.
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Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
HTB-125 Hs 578Bst 人正常乳腺細(xì)胞
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.
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Cryopreservation
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Complete growth medium, 95%; DMSO, 5%. Cell culture tested DMSO is available as ATCC Catalog No. 4-X.
HTB-125 Hs 578Bst 人正常乳腺細(xì)胞
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Culture Conditions
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Temperature: 37°C
Atmosphere: Air, 95%; Carbon dioxide (CO2), 5%
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