General Protocol for Freezing and Thawing Cells
Precaution:
· Cells are supposed to be contamination-free in the form of bacteria, yeast, or fungi.
· Mycoplasma testing should be performed prior to freezing.
· Freezing media depends on the cell line.
· One vial must be saved for testing the success of the freeze.
Materials:
Phosphate Buffered Saline (PBS)
Trypsin/EDTA solution
Tissue Culture Media
Cold Freezing Media (usually 10% dimethylsulfoxide, DMSO)
Labeled Cryovials (~3 per 100-mm plate for)
100-mm plate of the confluent cell
Freeze Procedure:
Pre-freeze
- Make sure your cells for free from any contamination; make sure you test a sample for mycoplasma using Mycoplasma testing kits.
- Trypsinize cells (standard protocol).
- Resuspend cells in media, transfer to a sterile centrifuge tube, centrifuge at 1000 RPM and 4°C for 3-5 min.
- Remove supernatant with sterile Pasteur pipette.
- Quickly resuspend the pellet by adding 1 ml freezing media per vial to be frozen.
- Aliquot 1 ml freezing media plus cells per vial, and place on ice.
- Freeze overnight at -80°C.
- Transfer vials to liquid N2 tank for indefinite storage.Post-freeze
- Remove a vial from the liquid N2 tank and follow the thaw procedure below to test the success of the freeze.
Thaw Procedure:
- Warm tissue culture media without selecting antibiotics to 37°C, label 100-mm tissue culture plate.
- Remove a vial from the liquid N2 tank and hold in 37°C water bath until sides are thawed, but center remains frozen. Do not shake the vial.
- Put the cells in a 15 ml vial and add a traditional cell culture medium gently from the top. Mix by gently inverting the vial
- centrifuge the tube at 300g for 5 min. remove supernatant and resuspend the pellet in 1 ml media
- Gently pour cells into the plate.
- Add 9 ml warm media dropwise to the partially frozen cells.
- Place plate in the incubator.
Article By: Team Science scriber
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