=================================================================
Freezing and thawing cells
=================================================================


Freezing cells 
-----------------
1. Make cryopreservation ("freezing") Media (should be fresh-ish, try not to use longer than 1 month)
   
   - Glycerol should be used if the cell type to be cryopreserved may be adversely or unwantedly affected by exposure to strong bi-polar compounds such as DMSO.
   - Important: The cryopreservatives, especially DMSO, must be used fresh each time. If you buy large volumes, transfer a small amount to another tube, wrap in aluminum foil (DMSO is light sensitive) and use the aliquot.
     Minimize opening and closing the reagent bottle. This is to avoid oxidization and/or absorbtion potentially toxic materials from the air.

=========================== ============= =====================================================================================================================
Materials                    Percent         Purpose
=========================== ============= =====================================================================================================================
FBS                          >20%            FBS binds toxic materials released if some cells are lysed during the freezing or thawing process.
DMSO or sterile glycerol     10%             Cryopreservative, prevents ice formation which destroys cells
=========================== ============= =====================================================================================================================
  
1. Trypsinize, centrifuge, and resuspend cells in growth media with FBS

   - Typically resuspend in 1-3 mL for a T75 or T185. 
   - *Optional*: count cells with a hemocytometer to know how many cells are frozen per cryovial.  

2. Add cell suspension and freezing media to cryovial in a 1:1 ratio (typically, 0.5mL resuspended cells, 0.5 mL freezing media)

.. tip:: For sensitive cells like MEFs, resuspend in FBS so that final conc. is 90% FBS and 10% DMSO

4. Label cryovial with name, date, passage number (same as the trypsinized cells), and approximate number of cells (either from hemocytometer or fraction of confluent flask (i.e. 1/6th T185))
5. Freeze cryovial at -80°C inside a styrofoam container. This will allow slow freezing as to not incur cell damage.
6. After approximately 24 hours transfer cryovial to liquid nitrogen.


Plating Cells from Frozen Stock
--------------------------------------------------------

See also :ref:`seeding and plating cells <seed-plating-cells>`


Materials 

- Frozen vial of cells (i.e MEFs or HEK293Ts)
- Growth Media (ex. DMEM with 10% FBS)

1. Rususpend contents of frozen vial with 10-20 mL of media and spin down at 400G for 4 minutes.  
2. Aspirate media to remove DMSO, careful to not aspirate the cell pellet.
3. Resuspend cell pellet in ~1 mL of growth media. 
4. Use 10 µL to count with the hemocytometer.

   - Number of cells in one corner * 10,000 = # of cells / mL 

5.  Coat T75 with 0.1% gelatin (required for MEFs, optional for HEK293Ts)

    - Add 5-7 mL of sterilized 0.1% gelatin to T75. 
    - Put T75 sideways and let sit at room temperature for minimum 10 minutes.
    - Right before use, aspirate gelatin liquid. 

6. Plate onto a T75 with 10 mL of growth media. Label flask with your initials, the date, cell type, and passage number (+1 the passage number label on the cryovial)
7. Incubate at 37°C. These cells will take 1-2 days to recover.