Jul 12, 2023

Public workspaceWater preservation for flow cytometry

DOI
dx.doi.org/10.17504/protocols.io.kqdg34eo7l25/v1
  • 1University of Michigan
Rachel Cable
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.kqdg34eo7l25/v1
Protocol Citation: Melissa B Duhaime, Rachel Cable 2023. Water preservation for flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg34eo7l25/v1
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
We use this protocol and it's working
Created: July 31, 2018
Last Modified: July 12, 2023
Protocol Integer ID: 14292
Abstract
This method of preservation for flow cytometry was adapted from the TARA Oceans protocol for bacterial and viral flow cytometry.

This version of the protocol was edited by Rachel Cable for use in EXPORTS 2018 cruises and for field work in Microbes in the Wild: Advanced Environmental Microbiology Lab (EEB 447) course at University of Michigan Biological Station and on campus.

Materials
MATERIALS
ReagentAQUEOUS GLUTARALDEHYDE 25% EMFisher ScientificCatalog #50-262-19
Protocol materials
ReagentAQUEOUS GLUTARALDEHYDE 25% EMFisher ScientificCatalog #50-262-19
Materials, Step 1
ReagentEMD Millipore Swinnex™ Filter Holders - Dia.: 25mmFisher ScientificCatalog #SX00 025 00
Step 2
Reagent20um Nylon Net FilterMerck Millipore (EMD Millipore)Catalog #NY2002500
Step 3
Glutaraldehyde preparation
Glutaraldehyde preparation
For 1.5 mL sample volume:
Aliquot 7.5 µL of 25% aqueous gluteraldehyde into each cryovial. Store aliquoted glutaraldehyde at 4°C until use.
ReagentAQUEOUS GLUTARALDEHYDE 25% EMFisher ScientificCatalog #50-262-19
Note
We sample for FCM in triplicate, as most samples can only be completely defrosted and analyzed once.
Filtration preparation
Filtration preparation
Clean Swinnex filter holders by soaking in 10% bleach for 1-24 hrs. Then rinse thoroughly with MilliQ water. Allow to dry completely or use immediately. Syringes can also be bleached in the same manner and reused to reduce plastic waste.
Safety information
Please rinse filter holders until there is no hint of bleach smell and then an additional rinse or two. Any residual bleach will interfere with the flow cytometry of the samples. We learned this from the sampling in 2018 and lost the first 1-2 of each triplicate that past through the Swinnex after it was cleaned.

ReagentEMD Millipore Swinnex™ Filter Holders - Dia.: 25mmFisher ScientificCatalog #SX00 025 00
Using sterilized forceps, place a 20 um nylon net filter in the filter holder and tightly screw the filter holder together.
Reagent20um Nylon Net FilterEmd MilliporeCatalog #NY2002500
Sample preservation
Sample preservation
Using a sterile 10-ml syringe (see bleaching protocol in Step 2 if syringe is being reused) and the prepared filter cartridge, aliquot 1.5 mL of your 20 um-filtered seawater sample into each cryovial. Invert to mix. Label your cryovials with a ultra fine point Sharpie marker.
Incubate at room temperature in the dark for 15 minutes.
Duration00:15:00 fixation
Snap-freeze the samples in liquid nitrogen. Store at -80°C.