TITLE: Refractive index adjusted imaging medium: Iodixanol (RI ~ 1.41 and 1.42) - Yeast
AUTHORS: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald

[DESCRIPTION]
This protocol describes the steps to prepare imaging medium for Saccharomyces cerevisiae with adjusted refractive index. This medium is optimized for fluorescence imaging by the reduction of auto-fluorescence through an abundance of Adenine [1] and the repression of of the Met-promoted pp7- CP expression [2].

[BEFORE_START]
Have the following solutions premixed:

Glucose 20% 500 ml solution:
Concentration: 200 g/l
mix 100 g Glucose in 500 ml deionized water (ddH2O)

Adenine 100x 100 ml solution:
Concentration 3 g/l
mix 0.3 g Adenine in 100 ml ddH2O

Methionine 200x 50 ml:
Concentration: 17.12 g/l
mix 856 mg into 50 ml ddH2O

[STEPS]
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
1. Compound medium for autoclave
SECTION: Preparation of 30ml refractive index adjusted imaging medium
1.1. Fill a 50 ml flask with 25 mLOptiPrep.
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 30ml refractive index adjusted imaging medium
1.2. Add 0.3 mLAdenine 100x solution.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium
1.3. Add 0.3 mLMethionine 200x solution.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium
1.4. Add 57.6 mg SC-XX powder. 
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium
1.5. Add 201.6 mg Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids (YNB).
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium
1.6. Add 1.4 mLddH2O.
SECTION: Preparation of 30ml refractive index adjusted imaging medium
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium
3. When the medium cooled down to around 80 °C add 3 mL sterile Glucose 20%.
SECTION: Preparation of 30ml refractive index adjusted imaging medium
4. The medium can be store at the bench for 2 to 3 months.
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
1.1. Fill a 50 ml flask with 22.5 mLOptiPrep.
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
1.2. Add 0.3 mLAdenine 100x solution.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
1.3. Add 0.3 mLMethionine 200x solution.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
1.4. Add 57.6 mg SC-XX powder. 
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
1.5. Add 201.6 mg Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids (YNB).
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
1.6. Add 3.9 mLddH2O.
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
3. When the medium cooled down to around 80 °C add 3 mL sterile Glucose 20%.
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Iodixanol
4. The medium can be store at the bench for 2 to 3 months.