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==MEF conditioned medium preparation== *'''Purpose:''' To prepare MEF conditioned medium for feeder free culture system *'''Batch #1''' **Date: 5/29/2012 ~ 6/13/2012 **Cells: MEFs from Dr. Yang Xu’s Lab; CF-1 MEFs from Global Stem (Cat# GSC-6001M, Lot# 94300663) ** Exp started on 5/29. MEF cells seed on 5/25 were used. Cell density: 2.5*10^4 cells/cm2. 10 wells. Collect MEF conditioned medium every 24 hrs (106 mL in total) from 5/30 to 6/4. Store at -20゚C. **Another exp started on 5/31. Thaw one vial of MEF (from Dr. Xu's Lab), seed in one T75 flask. Cell density: 4*10^4 cells/cm2. (Anticipated production: theoretically 0.3-0.4 mL/cm2). Collect MEF conditioned medium every 24 hrs (106 mL in total) from 6/2 to 6/8. Store at -20゚C. ** Another exp started on 6/4. Thaw one vial of MEF (CF-1 MITC treated, from GlobalStem) in one T75 and 2 wells of one 6-well plate. Cell density :~4*10^4 cells/cm2. Collect MEF conditioned medium every 24 hrs (106 mL in total) from 6/7 to 6/13. Store at -20゚C. **Pool all of the MEF conditioned medium and filter through a 0.22 um filter. Aliquot (40mL/tube) and store at -20゚C. Conditioned medium are good for one month. *'''Batch #2''' **Date: 7/24/2012 ~ 8/3/2012 **Cells: CF-1 MEFs from Global Stem (Cat# GSC-6001G, Lot# 94300639) ** Exp started on 7/24. Thaw 1 vial of CF-1 MEFs on to twenty 35mm-wells. Cell density: 2.5*10^4 cells/cm2. Collect MEF conditioned medium every 24 hrs (2*14=28 mL in total/day) from 7/25 to 8/3. Store at -20゚C. **Pool all of the MEF conditioned medium and filter through a 0.22 um filter. Aliquot (40mL/tube) and store at -20゚C. Conditioned medium are good for one month.
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