# Goal/Experiment:
To identify and count the population and relative size of the brown tide alga *Aureococcus anophagefferens* using flow cytometry techniques, specifically utilizing the Violet Side Scatter (SSC) channel on a Beckman Coulter CytoFLEX S Flow Cytometer.
# Aureococcus anophagefferens Population Count, and Relative Size (Violet SSC) by Flow Cytometry (CytoFLEX S Flow Cytometer Beckman Coulter)
### Authors
- Emily E. Chase
- Alex Truchon
- Steven W Wilhelm
*[The University of Tennessee, Knoxville](https://www.utk.edu)*
Published Dec 01, 2022
### DOI
[https://dx.doi.org/10.17504/protocols.io.q26g7yby9gwz/v1](https://dx.doi.org/10.17504/protocols.io.q26g7yby9gwz/v1)
### Keywords
- cytoflex
- flow cytometry
- violet side scatter
- cell size
- cell counts
- Aureococcus anophagefferens
### Abstract
A method for obtaining relative cell size, population density, etc., of the brown tide algae *Aureococcus anophagefferens* by a Violet Side Scatter (SSC) configuration on a Beckman Coulter CytoFLEX S Flow Cytometry System (CytExpert software).
### 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.
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## Guidelines
To achieve the intended results of this protocol, a configuration using a violet laser must be set up for a CytoFLEX S Flow Cytometer. This protocol is based on a CytoFLEX with Violet SSC on the violet laser at the 405/10 position. Refer to the "Setting Up Violet Side Scatter (VSSC) Channel" section of the CytoFLEX manual. Visualization of *A. anophagefferens* can be achieved without a violet laser, but it will not be possible to determine relative cell size.
## Materials
- **CytoFLEX S Flow Cytometer**: A flow cytometry device used to count and analyze particles suspended in a fluid.
- **CytExpert Software + desktop computer with appropriate specs**: Software used to control the CytoFLEX system and analyze data.
- **96 well CoStar Assay Plates [REF#3795]**: Plates used for sample preparation and analysis.
- **1000mL pipette and tips**: For transferring samples.
**Note**: Sheath fluid + cleaning fluid + Milli-Q water (or alternative) as required for cytometer maintenance only.
## Before Starting
Boot up the CytExpert Software and follow the start up protocol. Familiarity with the machine and its settings will permit necessary adjustments for your experiments and help ensure accurate results.
## Protocol
### CytoFLEX Experiment Setup
1. **Create a new experiment with sample acquisition settings as follows:**
- FSC 200
- SSC 40
- VioSSC 75
- FITC 200
- PerCP 150
- PB450 1 (placeholder; this could be set for a different dye than Pacific Blue shown here, this will not change the results)
- Threshold: (manual) PerCP 5000
Set the flow rate to medium (30 µL/minute), the sampling to 60 seconds (not by events), and the display to 100,000 events.
**Note:**
- Flow rate may need to be adjusted for a high abort percentage (>10%), both adjusting the rate (custom or otherwise) and diluting dense culture samples will solve this problem. Users should aim for a certain number of events per second, typically between 300–2000.
- Settings can be adjusted in real time by selecting "run" on samples and changing the acquisition settings.
2. **Optional**: Create a combination of density plots and histograms best suited for your analyses. An example set up is provided in the image below (Figure 2), where **A. anophagefferens** populations are clearly achieved.
- It is recommended to set up a histogram with time on the x-axis (final histogram) to account for the machine's measuring consistency as sampling will often start out slower and then stabilize.
3. **Sampling Process**:
- 250 µL of each sample to be measured (e.g., *A. anophagefferens* culture) are pipetted into a 96 well CoStar [REF#3795] Assay Plate round bottom plate (can be substituted) and loaded into the CytoFLEX.
- After opening the "Plate" window and clicking "Add Plate", samples can then be labelled.
**Note:**
- Volumes can be reduced after taking into account the flow rate and sample timing.
4. **Data Acquisition**:
- Labelled samples can then be run sequentially using the "Auto Record".
- Upon completion, data can be either exported (most conveniently as a .csv) or reviewed using the "Statistics" window according to all events and events within user defined gated populations.
- Population counts are achieved by number of events within the *A. anophagefferens* gates, and subsequent relative size of events (cells) can be achieved through Violet Side Scatter channel results.
**Note:**
- Relative size by Violet SSC cannot be achieved for every cell type (normally "larger" microalgae cannot), a priori comparisons with other cell size calculation methods (e.g., FlowCam) must be conducted.
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