Feb 17, 2020
Version 2
Marchantia high throughput imaging in multiwell plates V.2
- Marta Marta Tomaselli1,
- Marius Rebmann2,
- Mihails Delmans2
- 1University of Cambridge, Open Plant;
- 2Plant Sciences, University of Cambridge, OpenPlant
- OpenPlant Project
External link: https://github.com/HaseloffLab/MihailsDelmansThesis
Protocol Citation: Marta Marta Tomaselli, Marius Rebmann, Mihails Delmans 2020. Marchantia high throughput imaging in multiwell plates. protocols.io https://dx.doi.org/10.17504/protocols.io.bcmsiu6e
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: February 17, 2020
Last Modified: February 17, 2020
Protocol Integer ID: 33170
Abstract
This protocol allows high throughput imaging of Marchantia gemmae, using a cheap setup made with broadly available lab equipment. It was developed from the 3D-printed Imaging Growth Chamber developed by Mihails Delmans in his PhD work (https://github.com/HaseloffLab/MihailsDelmansThesis).
We used a transparent 384 wells plate filled with 1/2 strength Gamborg B5 media with 1.2% agar and placed a single gemma at the centre of the well. Gene frames and coverslips treated with anti-fog spray were used to cover (seal) the wells. This setup can be paired with automated imaging of samples.
Guidelines
Work under the flow hood to avoid gemmae contamination, but be aware that this causes the media to evaporate. To avoid it, try to cover the plate with a lid and leave it open for the shortest time possible while placing the gemmae.
Materials
MATERIALS
Gamborg B5 medium including vitamins Duchefa BiochemieCatalog #G0210
384 well microplate clear flat bottomgreiner bio-oneCatalog #781186
Gene Frame 1.7 x 2.8 cmThermo ScientificCatalog #AB0578
B-clean Anti-fog sprayCatalog #B250
Coverslip 22 x 50mmCatalog #0101142
Agar capsule 1gMelfordCatalog #1302310000
Before start
The set up we present in this protocol is optimised for our microscope stage travelling range and the size of available gene frames and coverslip. This does not mean that you cannot use larger areas of the plate.
Media preparation
Media preparation
Prepare 1/2 strength Gamborg media plus vitamin with 1.2% agar.
Plate set up
Plate set up
Pour the media into your selected wells, making sure they are filled up to the top and they not contain any air bubble inside.
Note
The number of wells to be filled depends on your requirements/ travelling range of the microscope stage.
We usually filled one more row/columns of wells around our samples to reduce media evaporation.
Let the media solidify and level it with a sterile scalple if it bulges out of the wells.
Place the gene frames to delimit the wells you are planning to image
Pick a single gemma from a gemma cup and place it at the centre of a well filled with media.
Note
Cover the plate with a lid to prevent media from evaporating when not adding gemmae.
Clean the cover glass with 70% EtOH and spray one side with the anti-fog spray.
Let the anti-fog solution evaporate and remove any residue with a lens tissue moving in one direction to avoid any visual artifacts.
Place the coverslip on the gene frame, making sure the anti-fog treated side is facing down.
The plate is now ready for imaging. Gemmae can be grown inside the plate up to 7 days.