Detecting reactive oxygen species in free-living symbiotic dinoflagellates exposed to nanoparticles

• 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich, CAS Number 4091-99-0, MW 487.29, Product Number D6883)

• Hydrogen peroxide (H₂O₂, 30%, Sigma-Aldrich, CAS Number 7722-84-1, MW 34.01, Product Number 216763)

• BD Difco^TM Marine Broth 2216 (dehydrated culture media, Fisher Scientific, REF 279110)

• Methanol (Fisher Chemical, A454-1, 0.2 micron filtered)

• MilliQ water

• Nanoparticle suspension

• Cell suspension (here Breviolum minutum , wild type, marine dinoflagellate algae)

• 15 mL conical tubes

• 50 mL conical tubes

• 1.5 mL or 2 mL Eppendorf tubes

• Multichannel pipettor (at least 8 positions) with 300μL volume/pipette

• 1 mL pipettor and tips

• 20-200 μL pipettor and tips

• 10-100 μL pipettor and tips

• 0.5 to 2.5 μL pipettor and tips

• 96 well dosing plate, round bottom, sterile

• 96 well plate, black, sterile (note: clear 96-well plate can also be used but a black plate will prevent bleed-through between wells)

• Troughs (reagent container)

• Aluminium foil

• Microplate reader

Dissolve 37.4 g of dehydrated BD Difco^TM Marine Broth 2216 in 1 L of MilliQ water.

Autoclave final solution.

Composition of BD Difco^TM Marine Broth 2216: https://www.fishersci.com/shop/products/bd-difco-dehydrated-culture-media-marine-broth-2216/DF0791174

Dilution 1: In a 2 mL Eppendorf tube, add 113 μL of stock solution of H₂O₂ and 886 μL sterile marine broth to achieve a final concentration of 1M H₂O₂.

Dilution 2: in a 2mL Eppendorf tube, add 30 μL of dilution 1 and 270 μL of sterile marine broth to achieve a final concentration 0.1M H₂O₂.

Stock solution: Dissolve 24.4 mg of DCFH-DA in 50mL methanol to make 1mM DCFH-DA concentrated stock solution.

This solution can be stored at -20°C for up to 4 months.

Working solution: in a 15 mL conical tube, add 100 μL of stock solution and 9.9 mL of sterile marine broth.

In a 15 mL conical tube, add 6 mL of cell suspension (at 1x10⁶ cells/mL) and 60 μL of DCFH-DA working solution. Wrap the tube in aluminium foil for 60 min dark incubation at room temperature. This is for half a 96-well plate at 150 μL per well (row B-G, columns 1-6)

After 60 min dark incubation, centrifuge the solution at 2000 rpm for 3 min at room temperature. Remove to supernatant and add 6 mL of fresh sterile marine broth. Plate cells in row B through G, columns 1 through 6 with 150 μL per well. Rows A and H (column 1 through 6) should be filled with 150 μL of sterile marine broth as blanks. Wrap plate in aluminium foil to prevent photoactivation or bleaching of DCFH, if you are not ready to dose the cells yet. Best practice would be to prepare the dosing plate during the DCFH-DA loading period so that you are ready to dose the cells as soon as the DCFH-DA loading is completed.

The nanoparticles tested here are composed of CeO₂ with a poly(acrylic acid) coating and DiI fluorescent labeling.

When synthesized, the colloid solution concentration was 1.3M.

To obtain the working solution:

• dilution 1: in an Eppendorf tube, add 1 μL of colloid solution and 999 μL of marine broth;

• dilution 2: in an Eppendorf tube, add 1 μL of dilution 1 and 999 μL of marine broth;

• dilution 3: in an Eppendorf tube, add 10 μL of dilution 2 and 990 μL of marine broth (final concentration 3.6 μM)

Prepare a nanoparticle working solution from your colloid solution (nanoparticles in suspension)to a final concentration of 1.3M. [Note: this protocol was developed for testing CeO₂ nanoparticles and their potential for scavenging ROS in Breviolum minutum (symbiotic dinoflagellate often associated with Aiptasia anemones and Acropora corals)]

Preparation of the dosing plate:

• in a round-bottom 96 well plate transfer 250 μL of complete sterile marine broth to column 1 to 6 in row B;

• H₂O₂ replicates:

  add 225 μL of sterile marine broth to columns 1 to 3 in row C;

  add 225 μL of sterile marine broth to columns 1 to 3 in row D;

  add 225 μL of sterile marine broth to columns 1 to 3 in row E;

  add 225 μL of sterile marine broth to columns 1 to 3 in row F;

  add 297 μL of sterile marine broth to columns 1 to 3 in row G;

  add 3 μL of H₂O₂ working solution (dilution 2) to columns 1 to 3 in row G;

  serial dilution: transfer 25 μL of wells G1-G3 to F1-F3 (using multichannel pipettor), repeat process from F1-F3 to E1-E3, from E1-E3 to D1-D3, from D1-D3 to C1-C3;

• CeO₂ replicates:

  add 150 μL of sterile marine broth to columns 4 to 6 in row C;

  add 125 μL of sterile marine broth to columns 4 to 6 in row D;

  add 125 μL of sterile marine broth to columns 4 to 6 in row E;

  add 125 μL of sterile marine broth to columns 4 to 6 in row F;

  add 250 μL of CeO₂ (3.6 μM in sterile marine broth) to columns 1 to 3 in row G:

  serial dilution: transfer 125 μL of wells G4-G6 to F4-F6 (using multichannel pipettor), repeat process from F4-F6 to E4-E6, from E4-E6 to D4-D6, and 100 μL from D4-D6 to C4-C6;

Centrifuge the 96-well plate with plated cells: 2000 rpm for 3 min at room temperature.

Remove supernatant (from columns 1 to 6, rows B to G) carefully using a fine tip plastic dropper pipette or standard pipettor;

[Be careful not to aspirate cells because the dinoflagellates do not attach]

Using a multichannel pipettor, transfer 150 μL from the dosing plate to the 96-well plate, well for well (from columns 1 to 6, rows B to G). Wrap the plate in aluminium foil until you analyze it in the plate reader. The analysis should be done directly after. The reaction starts as soon as you transfer the reagents from dosing plate to the plate with cells so the longer you wait the more of the reaction you miss.

Measure fluorescence of DCF using excitation/emission wavelengths of 485/530nm using a microplate reader at desired time points (every minute for up to 3H for kinetics or 1H for initial reaction);

Report peroxide production as the fluorescence intensity variation of treated cells relative to untreated cells;