C.

elegans Staining With Dihydroethidium & an Oxidative Stressor:

Protocol
1. Grow worms to young adulthood as normal on spread OP50 plates usually 3
days after hatching
a. One day longer for RB

2. Dissolve dihydroethidium fluorescent dye in DMSO to make the stock solution

3. Calculate the correct volume of dihydroethidium necessary to add OP50

spread plate to reach a concentration of 10 M

a. Using 10 mL agar plate for the volume

b. For example, I was taking from 10 mM DHE stock concentration, and so
(x mL) (10 mM) = 10 mL (10 M). In this equation, x = 10 L of 10mM
DHE for a 10mL agar plate
WARNING: The dye is both light sensitive and unstable in
solution. Keep out of light when possible, and only use dye in
solution up to a month after placing in solution. Would be
better to order in smaller quantities so that the dye only goes
into solution immediately before intended use
IF treating with DEM, perform steps 4 and 5. If treating with a drug,
perform step 6. Skip whatever steps are not being performed/are not
necessary. Steps 4-9 are to be done the day before the intended staining.
4. Calculate the correct volume of DEM necessary to add to OP50 spread plate
to reach your desired treatment concentration.
a. Using 10 mL agar plate for the volume
b. For example, I was taking from 400 mM DEM stock concentration, and
so
(x mL) (400 mM) = 10 mL (10 mM). In this equation, x = 250 L of 400
mM DEM for a 10mM DEM plate

5. On the day before the intended staining, add amount of DEM in a 2 mL

centrifuge tube, and add S. basal to reach about 750 L.

6. Calculate the correct volume of the intended drug necessary to add to OP50
spread plate (make sure to use correct volume of your plate) to reach
your desired treatment concentration.

7. On the day before the intended staining, combine the DHE, DEM, and
the drug in a 2 mL centrifuge tube (if you are treating with DEM), and pipette
onto spread OP50 plate.
a. For my concentration, 260 L DHE/DEM + 490 L S. basal

8. If treating with DEM, 234 uL of ethanol total should be added to control

(DEM untreated) plates (calculated for 10 mL plates)
a. This is the amount of ethanol present in 250 uL of 400 mM DEM
 9. Spread the solution around the plate so that the entire area of the plate is
covered. Place in a dark space overnight to ensure complete drying of the
plate.
10.Working with young adult C. elegans

a. Take 1 worm plate, and wash off young adult worms with about 2 mL S.
basal into a 2 mL microcentrifuge tube
b. Allow larger worms to settle, and pipette out supernatant
i. Getting rid of any eggs, younger worms that potentially hatched
late
c. Add 1 mL S. basal to the plate for an extra wash
d. Repeat steps B-C 2 times (2x wash)

11.Once worms have settled after 2nd wash, pipette out additional S. Basal, and
pipette worms onto the prepared (now dry) OP50 plate spread with DHE/DEM
(from steps 4 and 5)

WARNING: Be sure to not add too many worms to the plate,

particularly with N2. You dont need that many worms for your
experiment, and adding too many will cause starvation and
mess with your results (Shoot for 300-400)

12.Place the plates in a box in the 20C worm incubator, and allow to sit for a 24
hour period

IF treating with DEM, REPEAT STEPS 4-5. If treating with a drug, REPEAT
STEP 6. Skip whatever steps are not being performed/are not necessary.
STEPS 4-9 SHOULD BE REPEATED, AND ARE TO BE DONE THE DAY BEFORE
THE INTENDED STAINING. Make sure to prepare these plates WITHOUT THE
DYE.
- Repeat these steps on fresh OP50 plates. Make
sure to have enough OP50 plates before starting
the experiment.

13.After 24 hours, wash down the now treated adult worms with 2 mL S. basal,
and place in a 2 mL micro centrifuge tube

a. Repeat steps 10 B-C in order to wash any remaining dye away from the
worms

14.Pipette the worms onto the fresh OP50 plate prepared with the right
concentrations of DEM/drug, and leave for 1 hour
a. This is to clear any residual dye from the gut of the worms

15.After 1 hour, add 500 L of 4 mg/mL levamisole to the plate

a. This paralyzes the worms, making data collection with the Biosorter
more consistent
 b. After 2 minutes, use 8-10 mL of S. Basal to wash each treatment of
paralyzed worms into a 50 mL Eppendorf tube.

16.Samples now ready to analyze with the Biosorter (12 th floor)