Freezing and Thawing procyclic Trypanosomes
- Per strain to be frozen: make a solution of 1ml glycerol + 4ml media containing necessary drugs (5ml total).
- Aliquot 1ml into each of 5 cryovials.
- Add 1ml cells at 1-2x 106/ml concentration growing with 15% serum.
- Place vials in -80°C freezer 1 day.
- Transfer vials to liquid nitrogen the next day
- 1ml cells per vial.
- Add 0.2 ml glycerol, 0,8 ml additional medium.
- Cells should be fairly concentrated.
- Steps 4-5 as above.
- Per strain to thaw, aliquot 15ml falcon tube containing 8ml medium (SDM-79 10% or other).
- Remove a single vial from liquid nitrogen and set in 37°C bath until melted.
- Transfer the 2 ml contents to the 8ml medium in falcon tube.
- Pellet cells at 1500rpm for 10 minutes in swinging bucket rotor at room temperature.
- Remove supernatent, resuspend cells in 10ml medium and transfer to flask.
Download Protocol: Freezing and Thawing Trypanosomes
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