Aug 24, 2024
Refractive index adjusted imaging medium: Iohexol (RI ~ 1.4) - Yeast
- Mathias Hammer1,
- Ammeret Rossouw1,
- Azra Lari2,
- Ben Montpetit3,
- David Grunwald1
- 1UMass Chan Medical School, RNA Therapeutics Institute, Worcester, MA, USA;
- 2University of Alberta, Department of Cell Biology, Edmonton, AB, Canada;
- 3University of California, Department of Viticulture and Enology, Davis, CA, USA
Protocol Citation: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald 2024. Refractive index adjusted imaging medium: Iohexol (RI ~ 1.4) - Yeast. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5xxwg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 13, 2024
Last Modified: August 24, 2024
Protocol Integer ID: 103378
Keywords: yeast imaging, refractive index medium, yeast refractive index, Iohexol, live cell imaging
Funders Acknowledgement:
NSF
Grant ID: 1917206
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Abstract
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].
Materials
SC-Ura Powder
Sunrise Science Products
Cat#: 1306-030
Lot#: 23K3083
Exp: 10/2027
Yeast Nitrogen Base Without Amino Acids
Sigma Life Science
Cat#: Y0626-250G
Lot#: SLBG0555V
Glucose
Sunrise Science Products
Cat#: 1907-1kg
Lot#: 3A0036
L-Adenine
Sigma Life Science
Cat#: A-9795
Lot#: 33H12895
L-Methionine
Sigma Life Science
Cat#: M-5308
Lot#: 129H0322
Histodenz
Sigma Life Science
Cat#: D2158-100G
Lot#: WXBC3389V
Deionized Water
Equipment:
50 ml laboratory bottle with screw cap
1ml pipette
25 ml pipette
stirring hot plate
magnetic stirring bar
micro scales
autoclave
thermometer
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
Optional:
SC-xx 10x 100ml solution:
Concentration: 19.2 g/l
mix 1.92 g into 100 ml ddH2O
YNB 20x 100ml solution:
Concentration: 134.4 g/l
mix 13.44 g into 100 ml ddH2O
Compound medium for autoclave
Medium preparation with pre-resolved components9 steps
This version of the protocol shows the preparation of the medium from SC-XX 10x and YNB 20x solutions.
Fill a 50 ml flask with 21.9 mL ddH2O
Add a magnetic stirring bar and place the flask on a stirring hot plate.
Add 0.3 mL Adenine 100x solution.
Note
The additional Adenine is supposed to repress the Adenine synthesize to reduce a possible accumulation of red pigment [1].
Add 0.3 mL Methionine 200x solution.
Note
The additional Methionine represses the Met promoter, which drives PP7 syntheses [2].
Add 3 mL SC-XX 10x solution.
Note
In regard to cover all optional dropout media the amino acid base holds the notification -xx, where xx stand for the amino acid(s) that is as selection factor, missing in the medium.
Add 1.5 mL YNB 20x solution (Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids).
Add 36.519 g Histodenz (Iohexol) powder.
Note
Warming the medium on the stirring plate helps resolving the powders.
Autoclave for 00:15:00 at 121 °C .
Note
Remove the stirring bar before going to autoclave.
15m
When the medium cooled down to around 80 °C add 3 mL sterile Glucose 20%.
Note
The medium can be store at the bench for 2 to 3 months.
Protocol references
[1] Kokina, Agnese et al. "Adenine auxotrophy–be aware: some effects of adenine auxotrophy in Saccharomyces cerevisiae strain W303-1A." FEMS yeast research 14.5 (2014): 697-707.
doi:10.1111/1567-1364.12154
[2] Lari, Azra, et al. "Live-Cell Imaging of mRNP–NPC Interactions in Budding Yeast." Imaging Gene Expression: Methods and Protocols (2019): 131-150.
doi.org/10.1007/978-1-4939-9674-2_9