Fibroblast Cell Culture Protocol
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- Fibroblast Cell Culture Protocol
Protocol for subculturing and freezing fibroblasts
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Media DMEM- Invitrogen #11960-044 (high glucose without L-glutamine) 15% FBS Fetal Bovine Serum, Hyclone SV30014-03 (from Fisher-Sci) 1% (1x) Penicillin-Streptomycin (Invitrogen#15140-122) 1% (1X) L-glutamine (Invitrogen#25030-081) 200 |
Trypsin Trypsin EDTA C .25% (Invitrogen#25200-056) Hank’s Balanced Salt Solution HBSS- (Invitrogen#14170 (1X) (-) calcium Chloride, (-)magnesium chloride, (-) magnesium sulfate) DMSO for Freezing Cell Culture Grade DMSO (American Bioanalytical #AB03091) |
How to handle newly arrived cell cultures
- After unpacking, observe the cells under an inverted microscope to make sure they did well during transport. Place flask in a 37C incubator overnight. Do not remove medium.
- The next day, depending on the confluency of the cells, the flask may be fed by withdrawing the shipping medium and covering the cells with 5 mls of new medium (10 - 15% FBS) or subcultured according to the cell count.
Subculturing PRF cell lines
1) Cells should be split when confluent.
2) To split cells (volumes given are assuming cells are in a T25):
A. Using sterile technique, remove media and rinse flask with 2-3 mls of sterile HBSS. Remove and discard HBSS.
B. Add 1 ml trypsin and incubate for 2-3 minutes. Cells should be checked under an inverted microscope to determine if they have begun to round up, lift and float. If cells are not detached after 3 minutes, you may incubate another 1-2 minutes
C. Tighten cap and gently tip flask from side to side to dislodge all cells. Flask may be tapped lightly on side to loosen cells if needed.
D. Add 4 mls of new media to flask as soon as cells are floating to inactivate the trypsin. Rinse down sides of flask several times with the new media to wash all cells off the plastic and in to the solution. Remove all liquid containing cells and transfer to a 15ml conical (or 50 ml if you are pooling 3 or more flasks).
E. Using sterile technique, remove an aliquot for counting on a hemacytometer.
F. While you are counting cells, the rest of the solution should be spun in the clinical centrifuge for 5 minutes at 1000 rpms.
3) After calculating your cell count, plate cells at 250,000 cells per T 25 filter top flask.
4) Cells should be fed every 2-3 days with fresh growth medium.
Freezing:
Cells should be frozen at no less than 500,000 cells /ml/Corning freezer vial in 10% DMSO and subsequently placed in an isopropanol freezing chamber (Mr. Frosty) at -80 degrees C overnight and then transferred to the liquid nitrogen tank the next day.
For Further Information Please Contact:
Dr. Leslie Gordon
Medical Director, The Progeria Research Foundation
Assistant Professor of Pediatrics Research, Brown Medical School, Providence,
RI
Phone: 978-535-2594
Fax: 508-543-0377
Lgordon@progeriaresearch.org
Susan Campbell
Cell and Tissue Bank Coordinator
Phone (401) 863-7487
Fax (401) 863-3489
Susan_campbell@brown.edu