Measuring neutral lipids in fixed diatom cells using BODIPY 505/515
Phoebe Argyle, Jana Hinners, Sinéad Collins, Naomi M. Levine, Martina A. Doblin, Nathan G. Walworth
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.
Steps
Preparation of BODIPY stock solution
Add 2mg of 1mL of 2mg/mL .
Store this in dark glass at -20°C to prevent degradation of the fluorescent dye.
Diatom sample preparation
Sample the diatom culture of interest. For 'tube mode' flow cytometry take minimum 500µL into an eppendorf tube or other flow cytometry tube. For plate mode take 200µL into a round-bottom flow cytometry plate.
If using live cells, go to step 6
If using fixed cells:
Add 8% volume 10% (v/v) to reach a final concentration of approx 0.8% volume . For example, add 100µL of 1000µL of microalgae sample.
Agitate to mix, then leave to fix for 0h 10m 0s to ensure fixation
If samples are to be analyzed at a later date, samples should be stored in the fridge (max 48 hours), or flash frozen in -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
| Value | Label |
|---|---|
| CytoFLEX LX | NAME |
| Flow cytometer | TYPE |
| Beckman Coulter | BRAND |
| C40312 | SKU |
| 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 0.002mg/mL . For example add 2µL to 200µL of microalgae sample, or 2.2µL to a fixed microalgae sample (200µL sample with 20µL
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.
