Apr 03, 2017
Fluorescence activated cell sorting (FACS) of Perkinsus marinus transformants
- Protist Research to Optimize Tools in Genetics (PROT-G)
External link: https://doi.org/10.1038/s41592-020-0796-x
Protocol Citation: Imen Lassadi: Fluorescence activated cell sorting (FACS) of Perkinsus marinus transformants. protocols.io https://dx.doi.org/10.17504/protocols.io.hh2b38e
Manuscript citation:
Faktorová D, Nisbet RER, Robledo JAF, Casacuberta E, Sudek L, Allen AE, Ares M, Aresté C, Balestreri C, Barbrook AC, Beardslee P, Bender S, Booth DS, Bouget F, Bowler C, Breglia SA, Brownlee C, Burger G, Cerutti H, Cesaroni R, Chiurillo MA, Clemente T, Coles DB, Collier JL, Cooney EC, Coyne K, Docampo R, Dupont CL, Edgcomb V, Einarsson E, Elustondo PA, Federici F, Freire-Beneitez V, Freyria NJ, Fukuda K, García PA, Girguis PR, Gomaa F, Gornik SG, Guo J, Hampl V, Hanawa Y, Haro-Contreras ER, Hehenberger E, Highfield A, Hirakawa Y, Hopes A, Howe CJ, Hu I, Ibañez J, Irwin NAT, Ishii Y, Janowicz NE, Jones AC, Kachale A, Fujimura-Kamada K, Kaur B, Kaye JZ, Kazana E, Keeling PJ, King N, Klobutcher LA, Lander N, Lassadi I, Li Z, Lin S, Lozano J, Luan F, Maruyama S, Matute T, Miceli C, Minagawa J, Moosburner M, Najle SR, Nanjappa D, Nimmo IC, Noble L, Vanclová AMGN, Nowacki M, Nuñez I, Pain A, Piersanti A, Pucciarelli S, Pyrih J, Rest JS, Rius M, Robertson D, Ruaud A, Ruiz-Trillo I, Sigg MA, Silver PA, Slamovits CH, Smith GJ, Sprecher BN, Stern R, Swart EC, Tsaousis AD, Tsypin L, Turkewitz A, Turnšek J, Valach M, Vergé V, Dassow Pv, Haar Tvd, Waller RF, Wang L, Wen X, Wheeler G, Woods A, Zhang H, Mock T, Worden AZ, Lukeš J, Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nature Methods 17(5). doi: 10.1038/s41592-020-0796-x
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: March 31, 2017
Last Modified: March 16, 2018
Protocol Integer ID: 5402
Cell Culture and Electroporation
Cell Culture and Electroporation
See protocol "Oyster parasite Perkinsus marinus transformation using Amaxa electroporator and non-proprietary electroporation buffer"
Monitor transfection efficiency, by testing for the presence of fluorescent cells 5 to 6 days post transfection.
Cell recovery
Cell recovery
Once the transfection success is confirmed, transfer the cells to a larger volume in a T75 flask (or equivalent) to allow cell number to increase (up to one week).
Fluorescence activated cell sorting protocol
Fluorescence activated cell sorting protocol
The experiment should be undertaken in sterile conditions
Use Perkinsus marinus wild type cells as a control to set gating for non-fluorescent, single, live cells.
Set up a template that includes a bivariate plot to display forward scatter (FSC) and side scatter (SSC), and one histogram for each fluorophore that will be used (e.g. eGFP, mCherry)
Gate cells for fluorescence above that seen for the untransformed control cells, and sort these cells either as population or as single cells to a 96 well plate.
Sorted single cells in 96 well plates can then be cultured for 2 months at 25°C in the dark.
Inspect cells by microscopy to confirm fluorescence status.