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Fluorescence activated cell sorting in Schistosoma mansoni
Last updated date: Apr 28, 2020 View: 163
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See below for a detailed step-by-step protocol

 

Dissociation:

  1. Collect parasites in a 15mL conical tube

  2. Rinse parasites at least 2x with ~10mL of PBS

  3. Add 1.0mL Trypsin-EDTA (Sigma T4174) + 3mL PBS to each tube

  4. Pipette constantly with p1000 ~10m at RT

  5. Top both tubes with 8ml Basch media to stop digest

    • Can also use any media containing serum to inactivate the trypsin

  6. Spin 500rcf 10m at 4°C

  7. Resuspend each tube in 1mL Basch media

    • Can also use PBS+MgCl2/any media with MgCl2 (no EDTA)

  8. Filter through nylon mesh (100µM) to remove big chunks

    • Can also use a 100µM cell strainer

  9. Add 10µL/mL RQ1 DNase (Promega M610A)

    • This happens to be the one we use with in vitro transcription; it works, but there may be better/cheaper options

  10. Incubate 10m at RT

  11. Add 3mL PBS to each tube

  12. Spin 500rcf 10m at 4°C

 

DNA labeling:

  1. Resuspend each in 1 mL of Staining Media (see below) + 1uL of Hoechst(18mg/mL)

  2. Incubate 30m at RT in the dark

  3. Spin 500rcf 10m at 4°C

 

Final prep:

  1. Resuspend samples in 1mL Staining Media + 1uL Hoechst (18mg/mL in ddH2O stock), 1uL propidium iodide (1mg/mL in ddH­2O stock)

  2. Filter through nylon mesh (100µM) prior to sort, keep on ice

 

Sorting notes:

  • Sort all Hoechst positive/PI negative cells

  • Ask for FACS plots for gating strategies if you need

  • If your dissociation results in a great deal of debris, you might need to add a threshold parameter to the Hoechst signal such that the sorter only “sees” events that have an amount of Hoechst signal approximately equal to a nucleus’s worth of DNA.

  • If downstream application is single-cell RNAseq: sort into ~400uL of Staining Media w/o EDTA

    • Absolutely critical that there’s no EDTA

    • Automated cell counters will likely tell you that you have few/no live cells, so count yourself with hemocytometer

    • We resuspend the cells at 1000 cells/uL and then submitted the maximum volume of cells (I think 45uL of cells for 10x genomics V3 chemistry). Ask if you need more details.

 

Solutions

*Staining Media: PBS pH 7.40, 2mM EDTA, 0.5% BSA (MP Biomedicals 180620) 

  • You can adjust BSA concentration to between 0.1% and 1% and EDTA concentrations between 0mM and 5mM. Our recipe is a good starting point, but depending on your cells or your downstream applications you may wish to change some of the parameters.


Some other thoughts for optimizing the protocol on a different animal:


  • We dissociate using Trypsin in PBS but depending on the skin/cuticle/etc of whatever animal you're studying you may need to try different enzymes/buffers. The length of dissociation time may also vary a great deal, so at the beginning you'll probably need to carefully optimize this step.

  • Optimal centrifugation speeds/times may vary depending on the size, density, and fragility of the cells from your animal

  • We treat our cells with DNAse to help de-clump them because the dissociation protocol in schistosomes leads to a great deal of cell death and subsequent release of DNA from dead cells. You might not need this step depending on how well your animal tolerates being dissociated.

  • As noted in the protocol, our staining media is PBS, BSA, and EDTA. You may need to try different buffers/BSA concentration/EDTA concentrations, depending on your animal. Generally, the higher the BSA concentration, the happier the cells are, but the more autofluorescence you'll see. Adding EDTA up to 5mM helps prevent cells clumping together but can be bad for viability.

  • As noted in the protocol, when you get to the sorting step you may need to add a "threshold" to the Hoechst channel (essentially telling the sorter to ignore all events that don't have a minimum Hoechst signal approximately equal to one nucleus's worth of DNA). This is especially true if you have a great deal of debris from your dissociation. I can provide further help if you're working with a BD FACSAria-series sorter using the  FACSDiva software but I am not familiar with other cell sorters.

How to cite: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
1. Wendt, G. R.(2020). Fluorescence activated cell sorting in Schistosoma mansoni. Bio-protocol. bio-protocol.org/prep287.
2. Wendt, G., Collins, J., Pei, J., Pearson, M., Bennett, H., Loukas, A., Berriman, M., Grishin, N. and Collins, J.(2018). Flatworm-specific transcriptional regulators promote the specification of tegumental progenitors in Schistosoma mansoni. eLIFE . DOI: 10.7554/eLife.33221
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