Electroporation of fluorescent antisense molecules into Bodo saltans and live imaging

Abstract

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

Steps

Overview

Antisense peptide nucleic acids (PNAs, synthetised by Panagene, South Korea) were electroporated into Bodo slatans cells using Neon^® Transfection System MPK1025 (Invitrogen).

Bodo culture conditions

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.

Preparing cells for electroporation

The steps below describe how to prepare Bodo cells for electroporation.

3.1.

1-Filter the culture through 100 and 8 µm filter.

3.2.

2-Harvest the cells by centrifugation at 1200 × g for 12 mins at 19 °C.

3.3.

3-Wash the cells with 10 ml PBS and centrifuge as above.

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.

3.5.

5-Centrifuge at 1200 × g for 12 mins at 19 °C.

3.6.

6-Remove the PBS and resuspend the cells in resuspension buffer 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.

3.8.

8-Aspirate the mix into Neon pipette and electroporate using 1800 V, 10 ms pulse width and 1 pulse.

Preparing electroporated cells for imaging

The steps below describe how to prepare electroporated Bodo 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.

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).

4.3.

3-Wash the agarose with PBS (2×5mins) at RT.

4.4.

4-Remove the agarose from the well with clean forceps and place it on a microscope slide.

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.

4.6.

6-Proceed with either fluorescence or confocal imaging.

4.7.

Figure 1. Confocal image of liveBodo cell electroporated with antisense peptide nucleic acid (PNA). A) TAMRA-labelled antisense molecules inside Bodo cell. B) DNA of bacterial cells inside Bodo stained with Hoechst 33342.C) Overlay of two channels.

References

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