TITLE: Electroporation of fluorescent antisense molecules into Bodo saltans and live imaging
AUTHORS:   Mastaneh Ahrar, g.hurst  , Ewa  Chrostek

[DESCRIPTION]
This is the protocol used in our Laboratory at the University of Liverpool to electroporate molecules into Bodo saltans.

[STEPS]
SECTION: Overview
1. Antisense peptide nucleic acids (PNAs, synthetised by Panagene, South Korea) were electroporated into Bodo slatans cells using Neon® Transfection System MPK1025 (Invitrogen).
SECTION: Bodo culture conditions
2. Bodo saltans was cultured in a cerophyl-based medium enriched with 3.5 mM sodium phosphate dibasic (Na2HPO4)1. Cultures were incubated at 22 °C in T25 tissue culture flasks containing 20 ml of media bacterized with Klebsiella pneumoniae subsp. Pneumoniae (ATCC® 700831). 3 to 4 day old cultures were used for electroporation. 
SECTION: Preparing cells for electroporation
3. The steps below describe how to prepare Bodo cells for electroporation. 
SECTION: Preparing cells for electroporation
3.1. 1-Filter the culture through 100 and 8 µm filter.
SECTION: Preparing cells for electroporation
3.2. 2-Harvest the cells by centrifugation at 1200 × g for 12 mins at 19 °C.
SECTION: Preparing cells for electroporation
3.3. 3-Wash the cells with 10 ml PBS and centrifuge as above. 
SECTION: Preparing cells for electroporation
3.4. 4-Re-suspend the cells in 5 ml PBS, count the cells using hemocytometer and take the volume of cells which contains 5×10^5 cells, as recommended for Neon transfection kit for 10 µl tip.
SECTION: Preparing cells for electroporation
3.5. 5-Centrifuge at 1200 × g for 12 mins at 19 °C.
SECTION: Preparing cells for electroporation
3.6. 6-Remove the PBS and resuspend the cells in resuspension buffer for electroporation. 
SECTION: Preparing cells for electroporation
3.7. 7-Add the antisense molecule at the final concentration of 50 µM to the Bodo cells and mix well by pipetting. 
SECTION: Preparing cells for electroporation
3.8. 8-Aspirate the mix into Neon pipette and electroporate using 1800 V, 10 ms pulse width and 1 pulse.
SECTION: Preparing electroporated cells for imaging
4. The steps below describe how to prepare electroporated Bodo cells for imaging. 
SECTION: Preparing electroporated cells for imaging
4.1. 1-Empty the pipette of Neon system in a well of a 96-well plate after electroporation, add 5 µl of PBS, mix, add 15 µl of warm low melting temperature agarose (Thermo Fisher Scientific), and mix with the cells. Let is set for a few seconds. 
SECTION: Preparing electroporated cells for imaging
4.2. 2-Add 200 µl of Hochest 33342 (Thermo Fisher Scientific)diluted 1:2000 in PBSto the agarose-embedded Bodo and incubate 10 mins at room temperature (RT).
SECTION: Preparing electroporated cells for imaging
4.3. 3-Wash the agarose with PBS (2×5mins) at RT. 
SECTION: Preparing electroporated cells for imaging
4.4. 4-Remove the agarose from the well with clean forceps and place it on a microscope slide. 
SECTION: Preparing electroporated cells for imaging
4.5. 5-Add a drop of a mounting medium (eg. Vectashield, Vector Laboratories), and flatten the agarose as much as you can using the coverslip. 
SECTION: Preparing electroporated cells for imaging
4.6. 6-Proceed with either fluorescence or confocal imaging.
4.7.    

SECTION: References
5. Gomaa Fatma, Li ZuHong, Docampo Roberto, Girguis Peter, E. V. Bodo saltans culture protocol V.2. Protocols.io (2018). doi:10.17504/protocols.io.sh6eb9e