Feb 24, 2023
Measuring neutral lipids in fixed diatom cells using BODIPY 505/515
- Phoebe Argyle1,2,
- Jana Hinners3,
- Nathan G. Walworth4,
- Sinéad ollins5,
- 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
Protocol Citation: Phoebe Argyle, Jana Hinners, Nathan G. Walworth, Sinéad ollins, 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: November 30, 2020
Last Modified: February 24, 2023
Protocol Integer ID: 45067
Keywords: centric diatom thalassiosira spp, diatom cell, fixed diatom cell, measuring neutral lipid, phenotypic traits of microalgae, diatom plasticity, neutral lipid, frontiers in microbiology, throughput assay, microalgae, quantifying phenotypic trait, microbiology, cell
Funders Acknowledgements:
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
BODIPY™ 505/515 (4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene)Thermo FisherCatalog #D3921 Paraformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS)
Protocol materials
BODIPY™ 505/515 (4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene)Thermo FisherCatalog #D3921
Paraformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS)
Liquid nitrogen
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1435
Troubleshooting
Preparation of BODIPY stock solution
Add 2 mg of BODIPY™ 505/515 (4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene)Thermo FisherCatalog #D3921 in powder form to 1 mL of DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1435 to create the stock solution of 2 mg/mL .
Store this in dark glass at -20 °C to prevent degradation of the fluorescent dye.
Diatom sample preparation
10m
Sample the diatom culture of interest. For 'tube mode' flow cytometry take minimum 500 µL into an eppendorf tube or other flow cytometry tube. For plate mode take 200 µL into a round-bottom flow cytometry plate.
If using live cells, go to step 6
If using fixed cells:
Add 8 % volume Paraformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS) at 10 % (v/v) to reach a final concentration of approx 0.8 % volume . For example, add 100 µL of Paraformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS) to 1000 µL of microalgae sample.
Agitate to mix, then leave to fix for 00:10:00 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 Liquid nitrogen then stored at -80 °C .
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
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 1 % (v/v) to final concentration of approx 0.002 mg/mL . For example add 2 µL to 200 µL of microalgae sample, or 2.2 µL to a fixed microalgae sample (200 µL sample with 20 µL Paraformaldehyde 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.