TITLE: SRB viability assay for Acanthamoeba castellanii
AUTHORS: Carrie A. Flynn, Rebecca  Colon-Rios, Barbara Kazmierczak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Viability assay for </span><span style = "font-style:italic;">Acanthamoeba castellanii </span><span>trophozoites and cysts, which detects 1,000-100,000 cells. </span></div></div>

[STEPS]
?. [Cell preparation]
Grow Acanthamoeba castellanii trophozoites in  in vented tissue culture-treated 75 cm2 cell culture flasks at  without shaking. Passage 1:80 every 3-4 days.
[PYG media]
25 °C
?. [Cell preparation]
Add  to flasks and close lids tightly. Gently tap all surfaces of flasks on benchtop to dislodge trophozoites. Confirm complete detachment with light microscope at 100x. Move on to next step immediately, as trophozoites begin to reattach quickly (within 5 minutes).
[PYG media]
?. [Cell preparation]
Transfer media containing detached amoebas to a sterile 50-mL conical. Measure the volume using a sterile serological pipette and record (for cell count calculation).
?. [Cell preparation]
Briefly vortex conical to ensure even suspension of cells and transfer  to a 1.5-mL microcentrifuge tube containing . Mix by briefly vortexing and transfer  to hemocytometer.
100 µl
[trypan blue dye]
10 µl
?. [Cell preparation]
Briefly vortex conical of trophozoites to dislodge attached cells. Transfer appropriate volume of cell suspension containing desired number of trophozoites to a new conical. Pellet cells by centrifuging at  (in 50-mL conical) or  (in 15-mL conical).
Centrifuge: 1000 34
?. [Cell preparation]
Gently decant media into waste container and resuspend cell pellet by briefly vortexting. Adjust volume with fresh PYG to a final cell concentration of 106 per mL.
?. [Cell preparation]
Briefly vortex cells to dislodge attached trophozoites and ensure even suspension, then transfer to sterile liquid trough. Add  per well to sterile 96-well plates (clear, flat bottom, with lid).
[cells]
Plates must be tissue culture-treated for experiments on cysts.
?. [Cell preparation]
Allow plates to sit at  for approximately  before further processing to ensure cells have attached to plates. Before removing media for additional steps, spin plates  for .
0 Room temperature
Centrifuge: 200 34
Trophozoites are now ready for compound testing. To test compounds on cysts, proceed to steps 17 and 18.
?. [Compound preparation]
Dissolve compounds in appropriate vehicle to desired concentration. DMSO, methanol, and ethanol are nontoxic to A. castellanii  to a maximum of 1% final volume.
?. [Compound preparation]
Add dissolved compounds to media. For cysts, use EMb. For trophozoites, use LB or PYG. Trophozoites will continue to divide in PYG but not in LB (nor will they encyst).
?. [Cell preparation]
To induce encystment, remove media from wells and discard. Add  per well. Media must be added gently to walls of wells to prevent a jet of liquid from dislodging attached cells. Trophozoites can reattach, but cysts cannot.
[EMb encystment media]
(Do only if testing cysts)
?. [Cell preparation]
Remove spent PYG media from a monolayer of  trophozoites. Wash cells with , adding liquid to surface of flask opposite the cell layer to prevent dislodging adherent amoebas.
[PYG media]
?. [Cell preparation]
Count live cells and calculate total number of harvested amoebas. Determine total number of amoebas needed for experiment and calculate volume of cell suspension required.
?. [Compound testing]
Remove media from plates and discard. Add  per well, pipetting media gently to walls of wells to prevent a jet of liquid from dislodging attached cells.
[media +/- test compounds]
?. [Cell preparation]
Replace plate lid with breathable plate seal. Incubate at  without shaking for . At end of encystment, spin plates   for .
25 °C
Centrifuge: 200 34
(Do only if testing cysts)
?. [Compound testing]
Replace plate lid with breathable plate seal. Incubate at  without shaking for desired time.
25 °C
?. [Fixing plates]
At end of incubation, aspirate media and discard.
Alternatively, media can be removed by flicking off into waste container. Plates can also be washed with PBS-MC (either by pipetting or by submerging) before fixing, though this is not required for most test compounds. Plates can be fixed by submerging in TCA for faster processing.
If removing the media before fixing by flicking off into waste container, appropriate safety precautions must be taken as media may contain live amoebas—we suggest dumping plates into a wide basin containing bleach in a deep sink while wearing safety goggles, face shield, lab coat, and extended cuff gloves.
?. [Fixing plates]
Incubate plates for a minimum of  at . Plates can be stored at  for several weeks.
4 °C
4 °C
?. [Fixing plates]
Remove TCA by washing plates by submerging in plastic trays of tap water 4 times.
TCA is caustic. Appropriate PPE must be worn to prevent contact with skin or eyes.
Do not wash by running stream of tap water into plate wells. This results in uneven washing and can dislodge attached cells.
?. [Stain cells with SRB dye]
Dissolve SRB dye in  to a final concentration of 4% (w/v). For each 96-well plate, use  in .
[acetic acid]
[SRB dye]
5 mL
[acetic acid]
SRB dye is light sensitive and should be stored in the dark at  . We make it fresh for each experiment.
?. [Stain cells with SRB dye]
Add  per well and incubate plates at  for .
[SRB dye solution]
[in the dark]
SRB dye solution has high surface tension. Make sure it has covered entire well bottom before incubating. Gently shaking the plate can ensure the liquid has covered the well bottom.
?. [Stain cells with SRB dye]
Remove excess SRB dye by washing plates by submerging in plastic trays of  3 times. Gently shaking plates during submersion prevents air bubbles from excluding liquid, which can result in overstained wells. Contact time in acetic acid should be minimized to prevent overbleaching. Even washing of wells within and between experiments is critical for reproducible results. See video for example of technique.
[acetic acid]
We recommend changing first tray of acetic acid for each plate washed, and the second and third trays every other plate. This is because the majority of excess dye is removed with the first wash.
1% acetic acid is an irritant. Appropriate PPE must be worn to prevent contact with skin or eyes.
?. [Fixing plates]
Gently tap dry on paper towels and let air dry. Dried plates can be stored at  indefinitely.
0 Room temperature
?. [Stain cells with SRB dye]
Gently tap dry on paper towels and let air dry.
?. [Measure absorbance]
Solubilize SRB dye by adding  per well of .
150 µl
[Tris-HCl]
Precise pipetting is critical, as a change in volume will lead to a change in the concentration of solubilized SRB dye and the subsequent absorbance measurement.
?. [Measure absorbance]
Incubate plate on orbital rocking platform for  at .
0 Room temperature
?. [Measure absorbance]
Using a Kimwipe, remove any condensation from bottom of plate, top of lid, and inside lid. Measure the absorbance at 565 nm in a plate reader.
We use a Tecan Infinite M200 Pro plate reader with the following program: linear shake (1 mm amplitude) 30 seconds, wait 5 seconds, measure absorbance at 565 nm (25 flashes per read).
?. [Reagent preparation]
PYG mediumProtocol for preparation of PYG medium
?. [Reagent preparation]
EMb medium Protocol for preparation of EMb medium
Only needed if working with cysts
?. [Reagent preparation]
PBS supplemented with MgCl2 and CaCl2 (PBS-MC)
?. [Reagent preparation]
Make Solution A (20x, 500 mL): Na2HPO4 (final concentration of ) NaH2PO4 (final concentration of )    dH2O to
23.43 g
4.83 g
500 mL
?. [Reagent preparation]
Make Solution B (20x, 500 mL): CaCl2 (final concentration of ) KCl (final concentration of ) MgCl2 (final concentration of ) NaCl (final concentration of )    dH2O to
1.3 g
2.6 g
1.8 g
80 g
500 mL
?. [Reagent preparation]
Autoclave or filter sterilize solutions A and B. Store at .
0 Room temperature
?. [Reagent preparation]
Add  each of solutions A and B to  autoclaved dH2O for a final concentration of 1x PBS-MC. Store at .
50 mL
900 mL
0 Room temperature
?. [Reagent preparation]
Trichloroacetic acid (TCA): make 100% (w/v) TCA by adding  dH2O to  bottle of TCA. Dilute to  in 1x PBS-MC. Store in glass container at .
227 mL
500 g
4 °C
?. [Reagent preparation]
Prepare  acetic acid in dH2O in 10 or 20 L plastic carboy and store at .
0 Room temperature
?. [Reagent preparation]
Prepare  Tris-HCl  and store at .
0 Room temperature
?. [Fixing plates]
Add  per well. Add TCA gently to walls of wells to prevent dislodging attached cells.
[10% TCA in PBS-MC]