Read Lab: Researching Trypanosoma brucei | Protocols

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

Download Protocol: Freezing and Thawing Trypanosomes
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