TITLE: Quantifying Reactive Oxygen Species in diatoms
AUTHORS: Phoebe Argyle, Jana Hinners, Nathan G. Walworth, Sinéad Collins, Naomi M. Levine, Martina A. Doblin

[DESCRIPTION]
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.

[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. 


[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.

[STEPS]
SECTION: Initiation of assay
2. Remove algae cultures from growth conditions/incubator.

Transfer 2 x 500 µL  aliquots of microalgae culture into separate wells of a 48-well tissue culture plate. 
 

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

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

SECTION: Preparation of stain stock solution
1. Add 12.5mg oftto 5 mL   to create a 2.5 mg/mL stock solution. 

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

SECTION: Incubation
6. Wrap the whole plate in tin foil and incubate in the experimental conditions (return to culturing incubator) for 120 min 

 

SECTION: Analysis
7. 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. 

 


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. 
SECTION: ROS per well calculation
8. Calculate raw fluorescence (Relative fluorescence units RFU) for ROS with the following calculation:
Fluorescence of stained well - Fluorescence of the blank well

SECTION: ROS per cell calculation
9. 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). 

 


SECTION: Initiation of assay
4. Seal the plate with a Breathe-Easy sealing membrane to prevent evaporation during incubation. 
 


SECTION: Initiation of assay
5. Gently shake plate by hand to ensure even mixing of the stain within the culture.