Best practices for instrument settings and raw data analysis in plant flow cytometry

Abstract

Flow cytometry (FCM) is now the most widely used method to determine ploidy levels and genome size of plants. To get reliable estimates and allow reproducibility of measurements, the methodology should be standardized and follow the best practices in the field. In this article, we discuss instrument calibration and quality control and various instrument and acquisition settings (parameters, flow rate, number of events, scales, use of discriminators, peak positions). These settings must be decided before measurements because they determine the amount and quality of the data and thus influence all downstream analyses. We describe the two main approaches to raw data analysis (gating and histogram modeling), and we discuss their advantages and disadvantages. Finally, we provide a summary of best practice recommendations for data acquisition and raw data analysis in plant FCM.

A flow cytometry protocol for accurate and precise measurement of plant genome size using frozen material

“…Nuclei labelled with propidium iodide were excited by a blue laser (488 nm) and fluorescence was measured with a detector configured with a 695/40 nm bandpass filter on the Becton Dickinson LSR Fortessa X20 Cell Analyser. Fluorescence data was recorded on a linear scale of 256 channels (Koutecký et al, 2023). Leading trigger threshold was set to 5000.…”

“…Fluorescence data was acquired for 20 min at a low rate (12 µl/min) which delivered 10-20 events/s for fresh preparations but 100-150 events/s for frozen preparations. Postacquisition amplification of the signal was acquired by setting the forward scatter (FSC) detector voltage/gain to 320, side scatter (SSC) detector voltage to 179, and fluorescence detector voltage to 488 to position the internal standard peak at 1/5 th of the distance from the left end of the x-axis (Koutecký et al, 2023). Forward scatter and side scatter parameters were recorded on logarithmic scale and used to assist in…”

“…< 5%) was also considered for gating of the histograms (Loureiro et al, 2007b). In addition, minimum requirements for accurate GS estimation were followed as 2000 events in total and 600 events per peak (Koutecký et al, 2023). However, despite the lower event count, the estimation of GS was still pursued for comparative purposes, acknowledging that the data obtained may provide valuable insights and contribute to the broader understanding of variations between the two methods of nuclei isolation and data analysis.…”

“…During fluorescence measurements of the samples with moderate levels of debris, it is recommended to capture data of 2000 events in total and 600 per sample peak to maintain relative standard error (SE) below 0.2 % (Koutecký et al, 2023). However, the conventional method of nuclei isolation is highly sensitive to plant chemistry, buffer chemistry, and chopping style (Loureiro et al, 2021;Loureiro et al, 2006a, b).…”

“…However, the conventional method of nuclei isolation is highly sensitive to plant chemistry, buffer chemistry, and chopping style (Loureiro et al, 2021;Loureiro et al, 2006a, b). Despite optimisation of the conventional method, some plant species remain recalcitrant to the production of the required number of nuclei (Koutecký et al, 2023;Loureiro et al, 2021;Temsch et al, 2022). In particular, plant material high in secondary metabolites can be challenging and often requires substantially different buffers and chopping styles to generate a repeatable result (Čertner et al, 2022;Loureiro et al, 2006a;Noirot et al, 2000).…”