Bitan:Cell Culture

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Basic methods for PC-12 cells

Basic methods for culturing PC12 cells

 

PC12 is a cell line derived from a pheochromocytoma of the rat adrenal medulla.

 

Growth media formulation

Ham’s F12K media (2 mM glutamine) + 15% Horse Serum (heat inactived) + 2.5% Fetal Bovine Serum + 1% Antibiotic-Antimycotic (100X)

 

Differentiation media formulation

Ham’s F12K media (2 mM glutamine) + 0.5% Fetal Bovine Serum + 100 ng/mL NGF + 1% Antibiotic-Antimycotic (100X)

 

Heat inactivation of Horse serum

1. Remove horse serum from -80°C.

2. Sit in room temperature for 15 min and then thaw at 37°C.

3. When serum is thawed, place serum in 50°C water bath for 10 min (it is very important that it is only 10 min.)

4. Remove from water bath and make up media.

 

Starting cells

1. Warm growth media in 37°C water bath for about 20 min.

2. Take two 75cm2 flasks. Pipet 10 ml of growth media into each culture flask. Lay horizontally into the incubator for 15 min to adjust pH.

3. After 15 min, place the flasks into hood.

4. Take the frozen cells from liquid nitrogen tank.

5. Thaw cells in 37°C water bath in 1 min.

6. Pipet cells into 2 flasks.

7. Place the cells back into incubator.

8. Change media the second day.

 

Subculturing

1. Warm growth media in 37°C water bath for about 20 min.

2. Remove cells from incubator and check under microscope.

3. Remove culture media by aspiration with a glass pipette. Be careful. Don’t disturbing the cells.

4. Add 6 mL of media to each flask and rinse cells from flask by gently aspirating and dispensing media over the cells (Split 1 to 2-3 depending).

5. Add 2 mL or 3 mL of cells to new flasks with 8 mL of media.

6. Return flasks to incubator.

7. Clean hood with ethanol and wipe with kimwipes.

 

Freezing cells

1. Thaw freezing medium in 4°C and keep in 4°C until use.

2. Remove cell flasks form incubator. Remove culture media by aspiration with a glass pipette.

3. Collect cells with fresh pre-warmed growth media.

4. Count cells.

5. Calculate the volume of freezing medium needed to cell density 1×107~5×106 cells/mL.

6. Centrifuge cells at 200 g for 5-10 min. Remove media by aspiration with glass pipette.

7. Resuspend cells in 4°C calculated amount of freezing medium.

8. Fill 1 mL cells to each cryovial.

9. Transfer cryovials to freezing box.  The freezing box filled with 100% isopropylalcohol to the marked line.

10. Sit the freezing chamber in -80°C for 24 hours and then transfer to liquid nitrogen to store.

 

The method is modified from Dr. Erica A. Fradinger’s protocol.

 

More information about PC12 at: http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1721&Template=cellBiology

 

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