Feb 25, 2023

Public workspaceQuantifying Reactive Oxygen Species in diatoms

DOI
dx.doi.org/10.17504/protocols.io.ewov14qopvr2/v1
  • 1Climate Change Cluster, University of Technology Sydney, Ultimo, NSW, Australia;
  • 2Ministry of Marine Resources, Cook Islands;
  • 3Institute of Coastal Ocean Dynamics, Helmholtz-Zentrum Geesthacht, Geesthacht, Germany;
  • 4Department of Biological Sciences, University of Southern California, Los Angeles, CA, 90089-0371, USA;
  • 5Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, EH9 3JF, UK;
  • 6Sydney Institute of Marine Science, Mosman, NSW, 2088, Australia
Phoebe Argyle
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DOI: dx.doi.org/10.17504/protocols.io.ewov14qopvr2/v1
Protocol Citation: Phoebe Argyle, Jana Hinners, Nathan G. Walworth, Sinéad Collins, Naomi M. Levine, Martina A. Doblin 2023. Quantifying Reactive Oxygen Species in diatoms. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov14qopvr2/v1
Manuscript citation:
Argyle, P.A., Hinners, J., Walworth, N.G., Collins, S., Levine, N.M., Doblin, M.A., 2021. A High-Throughput Assay for Quantifying Phenotypic Traits of Microalgae. Frontiers in Microbiology 12(2910).
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: December 01, 2020
Last Modified: February 25, 2023
Protocol Integer ID: 45069
Keywords: Reactive oxygen species, phytoplankton, diatoms, ROS
Funders Acknowledgement:
Gordon and Betty Moore Foundation Marine Microbes Initiative
Grant ID: MMI 7397
Abstract
This protocol us designed to assess the relative concentration of reactive oxygen species in diatoms using a fluorescent dye.

This protocol is based on methods from:

Knauert, S., and Knauer, K. (2008). The role of reactive oxygen species in copper toxicity to two freshwater green algae J. Phycol. 44, 311-319.

Szivák, I., Behra, R., and Sigg, L. (2009). Metal‐induced reactive oxygen species production in Chlamydomonas reinhardtii (Chlorophyceae) J. Phycol. 45, 427-435.

This method was used in:

Argyle, P. A., Walworth, N. G., Hinners, J., Collins, S., Levine, N. M., & Doblin, M. A. (2021). Multivariate trait analysis reveals diatom plasticity constrained to a reduced set of biological axes. ISME Communications, 1(1), 59.

Argyle, P. A., Hinners, J., Walworth, N. G., Collins, S., Levine, N. M., & Doblin, M. A. (2021). A high-throughput assay for quantifying phenotypic traits of microalgae. Frontiers in microbiology, 12, 706235.
Image Attribution
Phoebe Argyle
Guidelines
This protocol has been developed using Thalassiosira spp. diatoms and may be applied to other taxa, however the staining concentration may need adjusting according to the permeability of cells to the dye.

As the incubation is performed in the dark, the effects of differential light treatments on cultures may not be captured.
Materials
ReagentH2DCFDA (H2-DCF, DCF)Thermo FisherCatalog #D399
ReagentDMSOP212121Catalog #GB-D-360
Equipment
Breathe-Easy® sealing membrane
NAME
Plate seal
TYPE
Breathe-Easy®
BRAND
Z380059-1PAK
SKU
https://www.sigmaaldrich.com
LINK

Equipment
Infinite® M1000 Pro
NAME
Microplate reader
TYPE
Tecan
BRAND
n/a
SKU
https://lifesciences.tecan.com/microplate-readers
LINK
Note M1000 Pro no longer in production, newer models such as the Spark are available and will serve the same purpose.
SPECIFICATIONS

Equipment
48 well Clear TC-treated Multiple Well Plates
NAME
Tissue culture plate
TYPE
Costar
BRAND
3548
SKU
https://ecatalog.corning.com/
LINK


Protocol materials
ReagentH2DCFDA (H2-DCF, DCF)Thermo FisherCatalog #D399
Materials, Step 1
ReagentDMSOP212121Catalog #GB-D-360
Materials, Step 1
Before start
Ensure your cultures are at the correct growth phase for measurement. We developed the protocol with cells growing in exponential growth but depending on the requirements of the researcher this may not be applicable.

Preparation of stain stock solution
Preparation of stain stock solution
1h
1h
Add 12.5mg ofReagentH2DCFDA (H2-DCF, DCF)Emd MilliporeCatalog #D399tto Amount5 mL ReagentDMSOEmd MilliporeCatalog #GB-D-360 to create a Concentration2.5 mg/mL stock solution.

When not in use store at Temperature-20 °C in the dark.

Initiation of assay
Initiation of assay
30m
30m
Remove algae cultures from growth conditions/incubator.

Transfer 2 x Amount500 µL aliquots of microalgae culture into separate wells of a 48-well tissue culture plate.
Equipment
48 well Clear TC-treated Multiple Well Plates
NAME
Tissue culture plate
TYPE
Costar
BRAND
3548
SKU
https://ecatalog.corning.com/
LINK

One well will act as a blank, the other as the treatment. Do this for all cultures being assayed.

Add Amount2 µL of 2’,7’dichlorodihydrofluorescein (H2DCF) stock solution from step 1 to the treatment wells as quickly as possible.

Seal the plate with a Breathe-Easy sealing membrane to prevent evaporation during incubation.
Equipment
Breathe-Easy® sealing membrane
NAME
Plate seal
TYPE
Breathe-Easy®
BRAND
Z380059-1PAK
SKU
https://www.sigmaaldrich.com
LINK


Gently shake plate by hand to ensure even mixing of the stain within the culture.
Incubation
Incubation
30m
30m
Wrap the whole plate in tin foil and incubate in the experimental conditions (return to culturing incubator) for Duration02:00:00

Note
As ReagentH2DCFDA (H2-DCF, DCF)Thermo FisherCatalog #D399 is a fluorescent dye and thus light-sensitive, any differences in light between culturing conditions may not be accurately reflected in this assay. E.g. a 'high light' vs. 'low light' treatment.


2h
Analysis
Analysis
Read the fluorescence of all wells (treatments and blanks) on a plate reader (e.g. Tecan Infinite® M1000 Pro) at excitation/emission of 488nm excitation 525nm emission.

Equipment
Infinite® M1000 Pro
NAME
Microplate reader
TYPE
Tecan
BRAND
n/a
SKU
https://lifesciences.tecan.com/microplate-readers
LINK
Note M1000 Pro no longer in production, newer models such as the Spark are available and will serve the same purpose.
SPECIFICATIONS


The complete list of settings that we used for our experiments were as follows:
Multiple reads per well 4x4 (circle, filled), border 1000 µm
Excitation wavelength: 488 nm
Emission wavelength: 525 nm
Excitation bandwith: 5 nm
Emission bandwith 5 nm
Gain: 100
Number of flashes: 50
Flash frequency: 400 Hz
Integration time: 20 µs
Lag time: 0 µs
Settle time: 10 ms

Gain settings will depend on the specific plate reader being used and on the density of the culture. If the culture is very dense the fluorescence will read as "OVER" in which case reduce the gain settings and read again. However, it is very important to maintain the same gain setting across an experiment in order to cross-compare different cultures.
ROS per well calculation
ROS per well calculation
30m
30m
Calculate raw fluorescence (Relative fluorescence units RFU) for ROS with the following calculation:
Fluorescence of stained well - Fluorescence of the blank well

ROS per cell calculation
ROS per cell calculation
1d
1d
Estimate the number of cells in each aliquot using flow cytometry (cite other protocol). Divide the value calculated in step 6 by the number of cells in the aliquot to obtain a ROS per cell measure (in RFU).

Note
If comparing between diatom taxa of different sizes, size correction may be applicable. In this instance forward scatter measures from the flow cytometer may be used as a proxy for cell size. A size correction can be done by dividing the ROS per cell measure from step 7 by the median forward scatter or the estimated cell size (when using size beads).