Quantifying Reactive Oxygen Species in diatoms
Phoebe Argyle, Jana Hinners, Nathan G. Walworth, Sinéad Collins, Naomi M. Levine, Martina A. Doblin
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.
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.
Steps
Preparation of stain stock solution
Add 12.5mg of5mL 2.5mg/mL stock solution.
When not in use store at -20°C in the dark.
Initiation of assay
Remove algae cultures from growth conditions/incubator.
Transfer 2 x 500µL aliquots of microalgae culture into separate wells of a 48-well tissue culture plate.
Equipment
| Value | Label |
|---|---|
| 48 well Clear TC-treated Multiple Well Plates | NAME |
| Tissue culture plate | TYPE |
| Costar | BRAND |
| 3548 | SKU |
One well will act as a blank, the other as the treatment. Do this for all cultures being assayed.
Add 2µL 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
| Value | Label |
|---|---|
| Breathe-Easy® sealing membrane | NAME |
| Plate seal | TYPE |
| Breathe-Easy® | BRAND |
| Z380059-1PAK | SKU |
Gently shake plate by hand to ensure even mixing of the stain within the culture.
Incubation
Wrap the whole plate in tin foil and incubate in the experimental conditions (return to culturing incubator) for 2h 0m 0s
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
| Value | Label |
|---|---|
| Infinite® M1000 Pro | NAME |
| Microplate reader | TYPE |
| Tecan | BRAND |
| n/a | SKU |
| 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
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
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).
