Extraction Of MEF Cells From Mouse Embryos And Treatment With Mitomycin C

The Extraction Steps Of MEF Cells From Mouse Embryos: 1. Put The Freshly Harvested Embryos Into A 10 Cm Cell Culture Dish. 2. Cover The Embryos With PBSA And Remove The Placenta And Other Maternal Tissues. 3. Cut Off The Top Of The Head (eyes And Above) And Remove The Viscera. 4. Save The Head/yolk Sac For DNA Separation For Genotyping. 5. Place The Embryos In A Separate 10 Cm Plate. 6. Add 1 ML Trypsin, Chop The Blade Into Small Pieces Of 1mm. 7. Disinfect The Blade Between Samples With An Alcohol Lamp Flame. 8. Place The Dish In An Incubator At 37 ° C For 30-45 Minutes (tilt It To Cover The Cells With Trypsin). 9. Suppress The Activity Of Typsin By Adding 4mL MEF Medium (usually DMEM+10% FBS+1% G/A) To Each Well. 10. Use A 10-20x Pipette To Blow And Separate The Tissue 11. Transfer The Cell Suspension To A T75 Flask Or A 10cm Plate, And Add 6-15mL MEF Medium. 12. Let The Cells Grow And Fuse (3-4 Days). 13. Extract The Medium, Wash It With 10mL PBS. 14. Add 3mL Trypsin, And Culture It In A 37 ° C Incubator For 5-10min. 15. Add 7mL MEF Medium For Neutralization. 16. Use A Pipette To Suck It Into A Centrifuge Tube For Centrifugation, So That The Cells Are Resuspended. 17. Transfer The Suspension To Two T175 Flasks, In Addition, 50 ML MEF Medium Was Added To Each Culture Bottle& Nbsp; Remarks: 1. Perform Cell Separation In Cell Culture Room And Pay Attention To Aseptic Operation& Nbsp;& Nbsp;& Nbsp;& Nbsp;& Nbsp; 2.  It Is Recommended To Use The Frozen Storage Solution Containing Serum, Such As Our Low Serum Immune Program Frozen Storage Solution, Qida Article Number: (SD0001) 3.  For Primary Isolation Culture, Please Use Gentamicin/amphotericin Solution To Prevent Cell Contamination. Qida Biological Product No.: SD0038. The Use Of Penicillin In P/S In The Medium Above 60ug/ml Will Lead To Antibiotic Resistance In The Later Stage Of The Cell, Thus Affecting Cell Transfection& Nbsp; MEF Inactivation Treatment/mitomycin C Treatment:; It Is Commonly Used In IPS Trophoblastic Cell Culture System. The Trophoblast Of Mouse Embryonic Fibroblast (MEF) Treated With Radiation Sterilization Or Mitomycin C Can Stop The Cell Cycle; This "inactivation" Prevents MEF From Growing Too Fast And Competing With Slower Growing PSCs& Nbsp; 1. Take A New Culture Bottle, Cover It With 0.1% Gelatin Solution For Pretreatment For 30 Minutes, And Suck Out Gelatin Solution Before Use. 2. Take MEF Cells To Be Treated, Add Mitomycin C At A Concentration Of 10ug/ml And Mix Well. 3. Put It In The Incubator For 3 Hours. 4. Suck And Discard Waste Liquid. 5. Wash With DPBS For 5 Times To Completely Remove Mitomycin C. 6. Add An Appropriate Amount Of Culture Medium To The Treated MEF For Resuspension Counting, With 3.0 × 104/cm2 (MEF) On Gelatin Treated Culture Bottle 7; Leave It In The Incubator Overnight To Make It Stick To The Wall 8; The Laid Feeding Layer Is Effective Within 1-7 Days, Which Can Support The Growth Of MES And Maintain Its Totipotency.