Thank you Eva Hatje... Here I have summarized below. Kind See and suggest me.
Method:
1. Place MEM and supplements into 37degrees Celsius water bath. Warm to temperature, approximately 30 minutes.
2. In laminar flow hood, assemble media filter and attach to house vacuum line.
3. Open line to start vacuum.
4. Add media to basin superior to filter. Add warmed supplements. Once all media and supplements are filtered, cap bottle and dispose of filter unit and centrifuge tubes used for aliquots in biohazard trash.
5. Label media bottle “MEM”, “20% FBS”, “1% P/S”, “1% Na Pyruvate”, date and initials.
6. Store media in refrigerator until time of use.
Cell Maintenance (Feeding Cells)to be done approximately every 2 days.
1. Heat media to 37 degrees Celsius in water bath.
2. In laminar flow hood, aspirate media with glass Pasteur pipet connected to house vacuum line.
3. Add 15 mL of media.
4. Tightly cap and return to 37 degrees Celsius incubator.
Passing Cells
1. Heat trypsin and media to 37 degrees Celsius in water bath.
2. In laminar flow hood, aspirate media with glass Pasteur pipet connected to house vacuum line.
3. Add 5 mL phosphate buffer solution (PBS) or 0.25% (w/v) Trypsin – 0.03% (w/v) EDTA to remove traces of FBS and residual Ca2+ that will deactivate EDTA. Immediately aspirate off solution.
4. Add 3 mL 0.25% (w/v) Trypsin – 0.03% (w/v) EDTA
5. Incubate for ~5 minutes at 37oC.
6. When cells have detached, add 7 mL media to quench trypsin. Reduce clumping by forcefully pipetting mixture against side of flask 4-5 times.
7. Pipette total volume into 15 mL centrifuge tube. Centrifuge for 10 minutes at 1.3 x 1000 rpm.
8. Remove supernatant.
9. Vigorously resuspend Caco-2 cell pellet with 4/6/10 mL media (for 1:4, 1:6, 1:10, respectively).
10. Add 14 mL to each new 75 cm2 flask.
11. Add 1 mL of Caco-2 cells to each flask.
Label and incubate at 37degrees Celsius. Feed cells by changing media approximately once every two days.
Thank you Eva Hatje... Here I have summarized below. Kind See and suggest me.
Method:
1. Place MEM and supplements into 37degrees Celsius water bath. Warm to temperature, approximately 30 minutes.
2. In laminar flow hood, assemble media filter and attach to house vacuum line.
3. Open line to start vacuum.
4. Add media to basin superior to filter. Add warmed supplements. Once all media and supplements are filtered, cap bottle and dispose of filter unit and centrifuge tubes used for aliquots in biohazard trash.
5. Label media bottle “MEM”, “20% FBS”, “1% P/S”, “1% Na Pyruvate”, date and initials.
6. Store media in refrigerator until time of use.
Cell Maintenance (Feeding Cells)to be done approximately every 2 days.
1. Heat media to 37 degrees Celsius in water bath.
2. In laminar flow hood, aspirate media with glass Pasteur pipet connected to house vacuum line.
3. Add 15 mL of media.
4. Tightly cap and return to 37 degrees Celsius incubator.
Passing Cells
1. Heat trypsin and media to 37 degrees Celsius in water bath.
2. In laminar flow hood, aspirate media with glass Pasteur pipet connected to house vacuum line.
3. Add 5 mL phosphate buffer solution (PBS) or 0.25% (w/v) Trypsin – 0.03% (w/v) EDTA to remove traces of FBS and residual Ca2+ that will deactivate EDTA. Immediately aspirate off solution.
4. Add 3 mL 0.25% (w/v) Trypsin – 0.03% (w/v) EDTA
5. Incubate for ~5 minutes at 37oC.
6. When cells have detached, add 7 mL media to quench trypsin. Reduce clumping by forcefully pipetting mixture against side of flask 4-5 times.
7. Pipette total volume into 15 mL centrifuge tube. Centrifuge for 10 minutes at 1.3 x 1000 rpm.
8. Remove supernatant.
9. Vigorously resuspend Caco-2 cell pellet with 4/6/10 mL media (for 1:4, 1:6, 1:10, respectively).
10. Add 14 mL to each new 75 cm2 flask.
11. Add 1 mL of Caco-2 cells to each flask.
Label and incubate at 37degrees Celsius. Feed cells by changing media approximately once every two days.
Hi Sanjay,
Another suggestion for filtering the media is to use sterile disposable syringes with sterile disposable syringe filter units and filter into sterile 50 or 15 ml centrifuge tubes. We found this to be very convenient. The amount of media you will require depends on the size of the flask that you are using, most come with a recommended maximum volume indicated on the flask. Good luck with your cell culture work!
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