Detecting nitric oxide in free-living symbiotic dinoflagellates exposed to nanoparticles

• Diaminofluorescein-2 diacetate (DAF-2 DA, C₂₄H₁₈N₂O₇, Sigma-Aldrich, CAS Number 205391-02-2, MW 446.4, SKU 251505-1MG-1)

• Sodium nitroprusside dihydrate (SNP, Na₂[Fe(CN)₅NO] · 2H₂O, Sigma-Aldrich, CAS Number 13755-38-9, MW 297.95, SKU 71778-25G)

• Sterile Calcium-Magnesium free seawater (CMFSW)

• 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

Combine

• 23 g/L NaCl

• 0.763 g/L KCl

• 3 g/L NaSO4

• 0.25 g/L NaHCO3

with DI water and autoclave

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 CMFSW;

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

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

Prepared a working solution of 3 mg/mL SNP in DI water (10 mM). Note: SNP is more soluble at >25°C.

Protect from light using aluminium foil

In a 15 mL conical tube, add 6 mL of cell suspension (at 1x10⁶ cells/mL in CMFSW) and 9 μL of DAF-2 DA stock 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)

Note: it is important to use CMFSW because DAF- 2 DA reacts with calcium ions in typical seawater

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 CMFSW. 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 CMFSW as blanks. Wrap plate in aluminium foil to prevent photoactivation or bleaching of DAF-2T, if you are not ready to dose the cells yet. Best practice would be to prepare the dosing plate during the DAF-2 DA loading period so that you are ready to dose the cells as soon as the DAF-2 DA loading is completed.

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 nitric oxide (NO) 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;

• SNP replicates:

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

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

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

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

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

  add 3 μL of SNP 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;

Avoid direct exposure to light.

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 DAF-2T using excitation/emission wavelengths of 495/515nm using a microplate reader at desired time points (every minute for 1H);