TITLE: Batrachochytrium dendrobatidis electroporation
AUTHORS: Andrew J. M. Swafford, Shane P. Hussey, Lillian K. Fritz-Laylin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Batrachochytrium dendrobatidis (BD) is a parasitic symbiote with many frogs. BD infection is devastating to most frog species, causing mass mortality in ~6.5% of amphibian species. To understand the cellular biology of BD and its interaction with amphibians, we have developed an method of electroporation, allowing for efficient deliver of extracellular constructs to infectious BD zoospores. The current method reliably loads 95% of treated cells with extracellular dextrans.</div></div>

[STEPS]
?. [Media & Material Prep]
Make 50 mL SM buffer (SM buffer should not be more than 7 days old).
?. [Media & Material Prep]
SM Buffer:
5 mL
[ KCL (Autoclaved)]
750 µl
[Sodium Phosphate Buffer (Filter Sterilized)]
7.5 mL
[MgCL2 (filter sterilized)]
[Sodium Succinate dibasic acid hexahydrate]
[D-Mannitol]
[Sterile water]
?. [Media & Material Prep]
Make an appropriate volume of Paraformaldehyde fixation buffer.
?. [Media & Material Prep]
Make 2mg/mL dextrans in SM buffer (Be sure it is Cat No. D3305 from Invitrogen by Thermo Fisher Scientific)        dextrans is sticky, measure in the tube you will be adding buffer to.        Spin down at high speed, then use supernatant.
?. [Media & Material Prep]
Measure out the required amount of autoclaved growth media into an appropriate flask.
?. [Media & Material Prep]
Place fixation buffer, growth media,  and bonner's salts on ice.
?. [Zoospore Prep]
Transfer synced cells to 50mL conicals
?. [Zoospore Prep]
Resuspend in 10 mL of SM buffer, combine after resuspension if multiple tubes were needed to hold all the synced spores.
?. [Zoospore Prep]
@  for  . Discard supernatant immediately.
Centrifuge: 2500 33
4 °C
?. [Media & Material Prep]
Turn Centrifuge on to
4 °C
?. [Zoospore Prep]
Resuspend in 5mL of SM
?. [Zoospore Prep]
Spin down cells and discard supernatant  @  for
Centrifuge: 2500 33
4 °C
?. [Zoospore Prep]
@  for  . Discard supernatant immediately.
Centrifuge: 2500 33
4 °C
?. [Zoospore Prep]
Resuspend cells in the appropriate volume of SM buffer needed for the # of cuvettes and tubes that will be used ( SM for each cuvette)
100 µl
?. [Zoospore Prep]
Count cell concentration. Aiming for 2x107 - 1x107 cells/mL
?. [Zoospore Prep]
If the spores are overly concentrated, dilute to the appropriate concentration.
?. [Zoospore Prep]
Prepare cuvettes & tubes.E- = electroporation control (no voltage)D- = Dextrans control (no dextrans)
?. [Zoospore Prep]
Prepare E-D- tubes. 1.5mL eppendorfs:
[SM]
[Cell suspension]
?. [Zoospore Prep]
Preprare E-D+ tubes: 1.5ml eppendorfs
[Dextrans solution]
[Cell suspension]
?. [Zoospore Prep]
Prepare E+D- cuvettes: 0.2cm electroporation cuvettes
[SM]
[Cell suspension]
?. [Zoospore Prep]
Prepare E+D+ cuvettes: 0.2cm electroporation cuvettes
[Dextrans solution]
[Cell suspension]
?. [Zoospore Prep]
Set samples on ice for ~ 10 minutes
?. [Electroporation]
Set electroporation protocol
?. [Electroporation]
Squarewave protocol1000V2 pulses5 sec intervals2mm cuvette
?. [Electroporation]
Insert cuvette
?. [Electroporation]
Hit the PULSE button, record percent droop
?. [Electroporation]
Remove cuvette and place immediately on ice.
?. [Electroporation]
[allow samples to rest on ice]
?. [Electroporation]
Add  to each cuvette
[chilled growth media]
?. [Electroporation]
Gently pipette liquid in cuvette up and down to mix cells just before electroporation
?. [Electroporation]
[allow samples to rest on ice]
?. [Wash cells]
Transfer contents of each tube/cuvette to 15mL conicals.
?. [Wash cells]
to each sample
[chilled growth media]
?. [Wash cells]
discard supernatant
Centrifuge: 2500 33
4 °C
?. [Wash cells]
Unless supernatant has been discarded three times.
?. [Wash cells]
add
[Bonner's salts]
[Bonner's Salts]
?. [Wash cells]
discard supernatant
Centrifuge: 2500 33
4 °C
?. [Wash cells]
until supernatant as been discarded 2 times
?. [Wash cells]
Aliquot  cells for imaging
100 µl
?. [Wash cells]
Aliquot   cells for counting
100 µl
?. [Wash cells]
Fix remaining cells for flow cytometry
?. [Electroporation]
Repeat steps 18-21 for all cuvettes, be sure to pipette control eppendorf tubes even though they will not be exposed to a pulse.
?. [Wash cells]
add
[Bonner's salts]
4 °C
?. [Cell Fixation]
Add an equal volume of fixation buffer (see  ) to each remaining volume of cells.
?. [Cell Fixation]
Immediately place tubes on ice.   (centrifugation time in the following step is IN ADDITION to this time). Leave alone for 15 minutes.
?. [Cell Fixation]
Centrifuge   @
Centrifuge: 2200 33
0 Room temperature
?. [Cell Fixation]
Properly dispose of supernatant and resuspend in
[Bonner's salts]
?. [Cell Fixation]
Immediately quantify via flow cytometry or wrap in foil and store at  for up to 4 hours.
4 °C
?. [Media & Material Prep]
Final Concentration of PFA fixation buffer:4.8% PFA9mM Sucrose50mM Sodium Phosphate Buffer, pH 7.2