Feb 25, 2023

Public workspaceMeasuring neutral lipids in fixed diatom cells using BODIPY 505/515

DOI
dx.doi.org/10.17504/protocols.io.4r3l2427jg1y/v1
  • Phoebe Argyle1,2,
  • Jana Hinners3,
  • Nathan G. Walworth 4,
  • Sinéad Collins5,
  • Naomi M. Levine4,
  • Martina A. Doblin1,6
  • 1Climate Change Cluster, University of Technology Sydney;
  • 2Ministry of Marine Resources, Cook Islands;
  • 3Institute of Coastal Ocean Dynamics, Helmholtz-Zentrum Hereon, 21502, Geesthacht, Germany;
  • 4Department of Biological Sciences, University of Southern California, Los Angeles, CA, 90089-0371, USA;
  • 5Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, EH9 3JF, UK;
  • 6Sydney Institute of Marine Science, Mosman, NSW, 2088, Australia
Phoebe Argyle
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DOI: dx.doi.org/10.17504/protocols.io.4r3l2427jg1y/v1
Protocol Citation: Phoebe Argyle, Jana Hinners, Nathan G. Walworth , Sinéad Collins, Naomi M. Levine, Martina A. Doblin 2023. Measuring neutral lipids in fixed diatom cells using BODIPY 505/515. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2427jg1y/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: December 01, 2020
Last Modified: February 25, 2023
Protocol Integer ID: 45067
Funders Acknowledgement:
Gordon and Betty Moore Foundation Marine Microbes Initiative
Grant ID: MMI 7397
Abstract
This protocol is for the measuring neutral lipids in fixed diatom cells using BODIPY 505/515, as developed on the centric diatom Thalassiosira spp.

This protocol has been used in the following publications:

Argyle, P. A., Walworth, N. G., Hinners, J., Collins, S., Levine, N. M., & Doblin, M. A. (2021). Multivariate trait analysis reveals diatom plasticity constrained to a reduced set of biological axes.ISME Communications,1(1), 59.

Argyle, P. A., Hinners, J., Walworth, N. G., Collins, S., Levine, N. M., & Doblin, M. A. (2021). A high-throughput assay for quantifying phenotypic traits of microalgae.Frontiers in microbiology,12, 706235.
Materials
ReagentBODIPY™ 505/515 (4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene)Thermo FisherCatalog #D3921 ReagentParaformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS)



Protocol materials
ReagentBODIPY™ 505/515 (4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene)Thermo FisherCatalog #D3921
Materials, Step 1
ReagentParaformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS)
In Materials and 3 steps
ReagentLiquid nitrogen
Step 5
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1435
Step 1
Preparation of BODIPY stock solution
Preparation of BODIPY stock solution
Add Amount2 mg of ReagentBODIPY™ 505/515 (4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene)Thermo FisherCatalog #D3921 in powder form to Amount1 mL of ReagentDMSOSigma AldrichCatalog #D1435 to create the stock solution of Concentration2 mg/mL .

Store this in dark glass at Temperature-20 °C to prevent degradation of the fluorescent dye.

Diatom sample preparation
Diatom sample preparation
10m
10m
Sample the diatom culture of interest. For 'tube mode' flow cytometry take minimum Amount500 µL into an eppendorf tube or other flow cytometry tube. For plate mode take Amount200 µL into a round-bottom flow cytometry plate.

If using live cells, go to step 6

If using fixed cells:

Add Concentration8 % volume ReagentParaformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS) at Concentration10 % (v/v) to reach a final concentration of approx Concentration0.8 % volume . For example, add Amount100 µL of ReagentParaformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS) to Amount1000 µL of microalgae sample.


Agitate to mix, then leave to fix for Duration00:10:00 to ensure fixation

10m
If samples are to be analyzed at a later date, samples should be stored in the fridge (max 48 hours), or flash frozen in ReagentLiquid nitrogen then stored at Temperature-80 °C .

Flow cytometry analysis
Flow cytometry analysis
Measure the background fluorescence of unstained cells using a flow cytometer. This protocol was developed using a CytoFlex LX.
Equipment
CytoFLEX LX
NAME
Flow cytometer
TYPE
Beckman Coulter
BRAND
C40312
SKU
https://www.beckman.com
LINK
CytoFLEX LX N3-V5-B3-Y5-R3-I2 Flow Cytometer (21 Detectors, 6 Lasers)
SPECIFICATIONS

Fluorescence is measured using 488 nm excitation and 525/40 nm detection.
Measure the background fluorescence of at least 200 cells but ideally 2000 or more.
Add the BODIPY stain in a Concentration1 % (v/v) to final concentration of approx Concentration0.002 mg/mL . For example add Amount2 µL to Amount200 µL of microalgae sample, or Amount2.2 µL to a fixed microalgae sample (Amount200 µL sample with Amount20 µL ReagentParaformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS) . Remember to take into account volume removed due to flow cytometry analysis.

Re-read the sample on the flow cytometer using the same laser parameters. Record the median fluorescence on the x channel for at least 200 cells

Calculate the change in median fluorescence between the stained and unstained sample. This is indicative of the level of neutral lipids present in the cell
If comparing between taxa of different sizes, size correction may be advisable. This may be done by dividing the change in median fluorescence by the equivalent spherical size, approximated from forward scatter which is measured in tandem during the flow cytometry analysis.