This post details the working protocol for PolyIC Seed tests using the resazurin metabolic assay.

Overview

This protocol is written for small oyster spat, but can be adapted for other life stages. This protocol is based on previous work in the lab by Colby E. and as used in our analysis of 10K Seed metabolic response and survival tests.

We are using this protocol to test metabolic response of seed oysters to thermal stress that are from parents that were immune primed using PolyIC exposure.

We will expose “treated” and “control” spat to our thermal stress protocol and will measure metabolic response using resazurin and will track survival in a 96-well plate format.

Materials and preparing solutions

1. Stock resazurin solution

To make the resazurin stock solution (10 mL) mix the following. We will use this solution for multiple trials.

• 0.5 g resazurin salt
• 10 mL DI water
• 10 µL DMSO

2. Working resazurin solution

To prepare the working solution of resazurin, prepare the following.

This recipe is to make 150 mL of working stock. To run a single 96 well plate, approximately 35 mL will be required (allowing for extra for re-do’s or errors). For example, if we are running 4 total plates, we will need to prepare 4 x 35 mL = 150 mL of working solution.

• 148 mL seawater (DI water with Instant Ocean adjusted to 23-25 ppt)
• 333 µL resazurin stock solution
• 150 µL DMSO
• 1.5 mL antibiotic solution 100x Penn/Strep & 100x Fungizone

3. Seawater

Prepare seawater solution using the FTR tank room hose water and Instant Ocean. Prepare one 500 mL bottles of water and put one on the counter at control temperature. These will be used for survival incubations at the end of the day after resazurin measurements.

4. Supplies

• Two Hobo TidbiT temperature loggers
  • One placed in incubator and one placed on counter at room temperature
• 96 well culture plates
• Paper towels and bench paper/pads
• Tweezers, transfer pipettes, and forceps
• Dissecting microscope
• Spectrophotometer plate reader(Perkin Elmer 1420 Multilabel Counter Victor3) and software version 3.00
• P1000 pipette
• Scale bar/ruler

Label plates with identifying number (e.g. “High Plate 1”, “Control Plate 2”).

Protocol

Resazurin measurements

1. Add 86 spat into a labeled 96 well plate (with a plate number and “resazurin”), all from the same treatment group. Repeat to generate 2-4 plates per treatment group, for a total of 4-8 96 well plates. Leave 10 random wells open for blank measurements.
2. Record plate number and position of samples in each plate. Assign plate number as “YYYYMMDD_TemperatureTreatment_Plate#”. For example, “20250110_High_Plate1”. Label plates consecutively, such that there is never a repeat of “plate 1”. Record which spat treatment group is in each plate and which wells contain oysters and which contain blanks.
3. Fill each well with 300 µL of resazurin working stock at ambient temperature.
4. Take a reading for each plate on the plate reader (initial time point). Save the file as “YYYYMMDD_TemperatureTreatment_Plate#_Time0.xlsx”.
5. Put the plates in respective temperature treatments. Put High plates at the 42°C incubation and Control plates at the countertop (~16°C).
6. After 1, 2, 3, and 4 hours, read the plates again on the plate reader. Record the name of data files as “YYYYMMDD_TemperatureTreatment_Plate#_Time1.xlsx” and so on.
7. Save the data to a flash drive and add to GitHub at the location here.

Survival measurements

1. After the 4 hour incubation, prepare new labeled plates with the same plate identification numbers and a label with “survival”.
2. Move the samples in the identical positions from the resazurin plate to the survival plate. In the transfer, gently rinse each spat by dunking into a clean seawater from the respective temperature to remove excess resazurin solution. Do this in a petri dish or cup.
3. While transfering, assess if the oyster is dead (gaping open with no movement) or alive (closed). Use a microscope if needed.
4. Record the plate, well number, and whether the oyster is dead or alive in the notebook or in a data sheet. You can do this by just making a list of the wells with either dead or alive spat.
5. Fill the wells of the survival plate with 300 µL seawater from the benchtop temperature.
6. Place all plates in the cold room on the table over night.
7. The next day (~24 h since start of resazurin measurements), record survival again as in step 4 above.
8. All oysters will be discarded after this time point.

Data sheets

Ariana will add links to data sheets here after initial trials.

Notes

Protocol is in progress and will be revised as testing is conducted.