Feb 25, 2023
Approximating silica uptake in diatoms using PDMPO and flow cytometry
- Phoebe Argyle1,2,
- Jana Hinners3,
- Nathan G. Walworth4,
- Sinead Collins5,
- Naomi M. Levine4,
- Martina A. Doblin1,6
- 1Climate Change Cluster, University of Technology Sydney, Ultimo NSW Australia;
- 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;
- 5Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK;
- 6Sydney Institute of Marine Science, Mosman, NSW
Protocol Citation: Phoebe Argyle, Jana Hinners, Nathan G. Walworth, Sinead Collins, Naomi M. Levine, Martina A. Doblin 2023. Approximating silica uptake in diatoms using PDMPO and flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9jpdqg3e/v1
Manuscript citation:
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(2910).
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: 45070
Keywords: diatoms, silica, PDMPO, biogeochemistry
Funders Acknowledgement:
Moore Foundation Marine Microbes Initiative
Grant ID: MMI 7397
Abstract
A protocol to quantify relative change in uptake of silica by diatoms using the proxy stain PDMPO.
This protocol is based on methods developed in:
Mcnair, H. M., Brzezinski, M. A., and Krause, J. W. (2015). Quantifying diatom silicification with the fluorescent dye, PDMPO.Limnol. Oceanogr. Methods13, 587–599. doi: 10.1002/lom3.10049
Mcnair, H. M., Brzezinski, M. A., and Krause, J. W. (2015). Quantifying diatom silicification with the fluorescent dye, PDMPO.Limnol. Oceanogr. Methods13, 587–599. doi: 10.1002/lom3.10049
Leblanc, K., and Hutchins, D. A. (2005). New applications of a biogenic silica deposition fluorophore in the study of oceanic diatoms.Limnol. Oceanogr. Methods3, 462–476. doi: 10.4319/lom.2005.3.462
Baker, K. G., Robinson, C. M., Radford, D. T., Mcinnes, A. S., Evenhuis, C., and Doblin, M. A. (2016). Thermal performance curves of functional traits aid understanding of thermally induced changes in diatom-mediated biogeochemical fluxes.Front. Mar. Sci.3:44. doi: 10.3389/fmars.2016.00044
This protocol is contained in:
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.
Image Attribution
P. Argyle
Materials
LysoSensor™ Yellow/Blue DND-160 Thermo Fisher ScientificCatalog #L7545
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
Equipment
48 well Clear TC-treated Multiple Well Plates
NAME
Tissue culture plate
TYPE
Costar
BRAND
3548
SKU
LINK
Equipment
96 Well TC-Treated Microplates
NAME
Microplate
TYPE
Corning®
BRAND
CLS3799-1EA
SKU
LINK
Equipment
Breathe-Easy® sealing membrane
NAME
Plate seal
TYPE
Breathe-Easy®
BRAND
Z380059-1PAK
SKU
LINK
Protocol materials
LysoSensor™ Yellow/Blue DND-160 Thermo Fisher ScientificCatalog #L7545
Materials, Step 1
Stock solution of PDMPO
Stock solution of PDMPO
Create a stock solution of PDMPO (2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole), also sold as LysoSensorTM Yellow/Blue DND-160, of 12.5 micromolar (µM) by diluting the 1 millimolar (mM) purchased solution in Milli-Q water.
LysoSensor™ Yellow/Blue DND-160 Emd MilliporeCatalog #L7545
Sample culture and fix for cell counts
Sample culture and fix for cell counts
Take a 200 µL aliquot of each experimental culture/well (if growing in well-plates) and transfer into a 96 well round-bottomed plate to count via flow cytometry (see step 6).
Equipment
96 Well TC-Treated Microplates
NAME
Microplate
TYPE
Corning®
BRAND
CLS3799-1EA
SKU
LINK
Initiate the assay
Initiate the assay
Transfer 2 500 µL aliquots of culture into a 48-well tissue culture plate. One to act as a control, the other to be treated with PDMPO.
Add 5 µL of the PDMPO stock made in step 1 to the treatment wells. This results in a final concentration of 0.125 micromolar (µM)
Seal the plates with a sealing membrane and agitate gently to ensure even mixing of the stain.
Equipment
Breathe-Easy® sealing membrane
NAME
Plate seal
TYPE
Breathe-Easy®
BRAND
Z380059-1PAK
SKU
LINK
Initiatial cell counts and background fluorescence
Initiatial cell counts and background fluorescence
Using a flow cytometer, count the concentration of the culture at T0 from the fixed aliquot using the protocol outlined below.
Protocol
NAME
Measuring growth rates of diatom cells in cultureCREATED BY
Phoebe Argyle
End the assay
End the assay
24 hours after the intiation of the assay, remove plates from the incubator and aliquot 200 µL of each culture (stained and unstained) into a round-bottomed 96 well plate for flow cytometry. Use a scalpel blade to slit the plate seal over each well.
Equipment
96 Well TC-Treated Microplates
NAME
Microplate
TYPE
Corning®
BRAND
CLS3799-1EA
SKU
LINK
Measure the PDMPO fluorescence of the stained and unstained cells using the flow cytometer, measuring at least 200 cells. PDMPO fluorescence is quantified using the near UV channel (275 nm excitation/490-530 nm emission).
Also record the cell concentration.
Calculations
Calculations
For the control samples:
Measure the median PDMPO fluorescence of at least 200 cells for each sample using the same method as in step 8.
Calculate the growth rate of the unstained cells over 24 hours using the equation:
where N is the number of cells per mL at time 2 (24 hours) and time 1 (initiation) and t2 and t1 are 1 and 0 (time in days).
For the stained samples:
Measure the median PDMPO fluorescence of at least 200 cells for each sample using the same method as in step 8.
Calculate the difference in median fluorescence between the stained and unstained cells after 24 hours.
To calculate the metric, use the following equation:
Note
Note, sometimes a double peak is observed during the flow cytometry measurements as cells divide (see image). This is accounted for by using the median fluorescence measurement.