Nov 26, 2024
Version 1
Yeast Sample Preparation for Fluorescence Live Cell Imaging V.1
- Mathias Hammer1,
- Ammeret Rossouw1,
- Brain Tran1,
- Anyee Esther Li1,
- Graham Latino1,
- pieterfop.vanvelde1,
- 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, Brain Tran, Anyee Esther Li, Graham Latino, pieterfop.vanvelde, Azra Lari, Ben Montpetit, David Grunwald 2024. Yeast Sample Preparation for Fluorescence Live Cell Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l629xdgqe/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: May 14, 2024
Last Modified: November 26, 2024
Protocol Integer ID: 99800
Keywords: yeast live cell imaging, yeast sample preparation
Funders Acknowledgements:
NSF
Grant ID: 1917206
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Abstract
This protocol describes the preparation of yeast cells for fluorescence single molecule tracking live cell imaging. This protocol utilizes linked protocols for specifics on media preparation. Special attention is given to creating single layer yeast suspensions on the cover glass.
Materials
Concanavalin A (ConA) from Canavalia ensiformis:
Sigma-Aldrich
Cat#: C2272-10MG
Lot#: 129H0322
UltraPure Distilled Water
Cover slides ⌀ 25mm, No. 1.5 or 1.5H
Attofluor Cell Chamber
Thermo Fisher Scientific
Cat#: A7816
Equipment:
10 ml centrifugation tube
10 ml pipette
Spectrometer: Molecular Devices SpectraMax m5
Plasma cleaner: Quorum Emitech K100X glow discharger
mixer
bench top centrifuge
Stock sample preparation
Stock sample preparation
2d 12h
2d 12h
Plate the yeast strain of interest onto pre-imaging solid agar plate.
Pre-Imaging Solid Growth Medium - Yeast protocol: https://dx.doi.org/10.17504/protocols.io.8epv5r9wjg1b/v1
Incubate at 25 °C until colonies are visible. (~2-3 days 60:00:00 ).
Note
Temperature and duration depending on the specific yeast strain.
2d 12h
The agar plate with grown yeast can be stored, sealed in 4 °C fridge, and can be used as stock for 2-3 months.
Preparation for imaging the next day
Preparation for imaging the next day
12h
12h
Pick a colony from your stock sample and dilute it in 5 mL liquid pre-imaging medium in a test tube.
Note
Choose the pre-imaging medium according to your labeling system. If it includes labeled pp7-stem loop (pp7-SL), its expression can be suppressed through higher Methionine concentrations in the medium [2]. A recipe for such a medium provides the protocol below that note.
For otherwise labeled yeast it is recommended to use media with higher concentrations of Adenine to reduce autofluorescence [1]. You can follow the "Pre-Imaging Liquid Growth Medium":
Pre-Imaging Liquid Growth Medium for Methionine promoted Labeling Systems - Yeast protocol:
Incubate at 200 rpm, 25°C Overnight .
Note
The incubation temperature depends on the yeast strain!
12h
Yeast conditioning
Yeast conditioning
4h 5m
4h 5m
Dilute the cultured yeast to OD600 = 0.25 - 0.3 and incubate 200 rpm, 25°C, 03:00:00 - 04:00:00 . Let the sample grow to an OD600 of 0.8 -1.0 to establish exponential growth phase.
4h
Sample preparation
Sample preparation
4h 5m
4h 5m
Pipette 1.5 mL of the incubated yeast into an Eppendorf-tube.
Centrifuge at 560 x g, 23°C, 00:05:00560
5m
Remove supernatant and re-dilute the pellet in 750 µL pre-imaging medium.
Plate the re-diluted cells into a cell chamber with a ConA prepared cover slide.
Protocol for the ConA cover slide preparation:
Cover the cell chamber with Kin wipe and let the cells settle in an incubator at 25 °C for 00:10:00 .
Note
Incubator temperature depends on the Yeast strain and can differ in respect to the experiment.
10m
Sample
Sample
10m
10m
Remove the supernatant with a 1 ml pipette.
Tilt the chamber slightly and add 1 mL of medium from the top corner. Remove the medium at the bottom corner to remove loose cells.
Repeat the last step to remove the second layer of cells.
Continue washing the sample as described, until you expect a single layer of cells in the central area of microscope slide.
Note
The pressure to remove the cells from the cover slide respectively the second layer of cells differs between yeast strains and the age (~use cycles) of the ConA.
Note
The removal of the second layer of cells reduces background fluorescence and increases the probability to have the nuclei in the same plan.
Space around the cell groups allows the yeast to grow in xy direction and less into z direction creating a new second layer, which allows for longer observation duration.
Cautiously add1 mL refractive index adjusted imaging medium (RI=1.41).
Refractive index adjusted imaging medium: Iodixanol (RI ~ 1.41 and 1.42) - Yeast:
Note
The ideal refractive index for the imaging medium depends on your experimental setup, especially on the immersion medium.
The RI of the medium can be adjusted by varying the RI gradient substance (here Iodixanol). Examples:
Place the the chamber in the stage incubator. Wait until the temperature reached equilibrium before you start imaging.
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