Oct 20, 2015

Public workspaceFixation of marine samples for flow cytometry sorting

 Forked from Fixation of marine samples for flow cytometry analysis
DOI
dx.doi.org/10.17504/protocols.io.d2x8fm
  • Daniel Vaulot1
  • 1Station Biologique, Roscoff, France
  • Ecology of Marine Plankton (ECOMAP) team - Roscoff
    Tech. support email: phytopk@sb-roscoff.fr
Daniel Vaulot
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DOI: dx.doi.org/10.17504/protocols.io.d2x8fm
Protocol Citation: Daniel Vaulot 2015. Fixation of marine samples for flow cytometry sorting. protocols.io https://dx.doi.org/10.17504/protocols.io.d2x8fm
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: October 20, 2015
Last Modified: March 27, 2018
Protocol Integer ID: 1847
Abstract
Protocol to fix marine samples for flow cytometry sorting of phytoplankton.
Fix at least 2 samples per depth sampled and at least 6 to 10 depths per vertical profile.

Back to the laboratory, pico and nano-plankton populations can be sorted and used for clone library construction or metabarcoding with Next Generation Sequencing (e.g. Illumina).  This was successfully tested on samples from a cruise off Brazil (unpublished data)

Reference
Ribeiro C., Lopes A., Marie D., Vaulot D. Unpublished data
Materials
MATERIALS
ReagentPluronic 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5556-100mL
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
STEP MATERIALS
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
ReagentPluronic 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5556-100mL
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
ReagentPluronic 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5556-100mL
Protocol materials
ReagentPluronic 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5556-100mL
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
ReagentPluronic 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5556-100mL
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
ReagentPluronic 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5556-100mL
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
ReagentPluronic 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5556-100mL
Safety warnings
Samples must be stored either in liquid nitrogen or at -80°C, not at -20°C because degradation will take place at the latter temperature
Before start
Necessary equipment
- Vortex mixer
- Cryotubes 2 mL
- Liquid nitrogen tank
Prefilter seawater sample onto 200 µm mesh
Amount2 mL
Add 1.5 mL of prefiltered seawater sample to a 2 mL cryotube
Add DMSO
Amount150 µL
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
Add Pluronic (facultative)
Amount2 µL
ReagentPluronic 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5556-100mL
Vortex
Wait 10 min
Duration00:10:00
Flash freeze in liquid nitrogen tank