The ploidy level of the 55 accessions (each analyzed in three biological replicates) was studied through flow cytometry (CyFlow Ploidy Analyzer) of 4’,6-diamidino-2-phenylindole (DAPI)-stained nuclei following the procedure described by the CyStain UV Precise P protocol (Sysmex Partec). One hundred milligrams of fresh leaf tissue taken from each of the three biological replicates were co-chopped with a razor blade in a Petri dish with 0.5 ml of Nuclei Extraction Buffer (Sysmex Partec) and incubated for 45 min at room temperature. After filtering (30 μm of CellTrics®, Sysmex Partec), 2 ml of staining buffer was added to each sample and incubated for 60 s before analysis (Nd-YAG green laser: λ = 532 nm; 30 mW, flow rate of 4 μl/s). Fluorescence histograms were evaluated using FCS Express 5 Flow software (Sysmex Partec), and ploidy levels were inferred by comparing each sample with the ploidy level of the 2001 sample.
Fcs express 5 flow software
Manufactured by Sysmex
Sourced in Germany
FCS Express 5 Flow is a software suite designed for the analysis and visualization of flow cytometry data. It provides a comprehensive set of tools for data management, gating, and statistical analysis.
Automatically generated - may contain errors
Lab products found in correlation
Celltrics, by Sysmex (3 mentions)
Nuclei extraction buffer, by Sysmex (2 mentions)
Cyflow ploidy analyzer, by Sysmex (1 mentions)
Cystain uv precise p, by Sysmex (1 mentions)
Cystain uv precise p nuclei extraction buffer, by Sysmex (1 mentions)
Cystain pi absolute p, by Sysmex (1 mentions)
Cystain pi absolute p nuclei extraction buffer, by Sysmex (1 mentions)
4 protocols using fcs express 5 flow software
1
Ploidy Analysis of Plant Accessions
2
Leaf Nuclei Extraction and Flow Cytometry
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Nuclei were isolated from 100 mg leaf tissue by gentile chopping with a razor blade in mL of CyStain UV Precise P nuclei extraction buffer (Sysmex Partec GmbH, Gorlitz, Germany) supplemented with 1% w/v PVP. For each considered sample, 3 replicates were analysed. Following nuclei extraction, the suspension was filtered through nylon tissue of 30 mm mesh width as recommended by the manufacturer. Following the filtration step, mL of staining buffer was added to the lysate and the tubes were stored in the dark on ice for 1 h before measurement. The fluorescence intensity of DAPI-stained nuclei was determined using the flow cytometer CyFlow Cube Ploidy Analyser (SysmexPartec GmbH, Gorlitz, Germany) equipped with an UV-Light Emitting Diode (l = 355 nm–375 nm). Data were plotted on a logarithmic scale and calibration of C values was made with nuclei extracted from C. limon. Ploidy histograms were quantitatively analysed with the FCS Express 5 Flow software (SysmexPartec GmbH), after manual treatment to exclude noise.
3
Genome Size Determination by Flow Cytometry
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The genome size of the “Mandevilla 2001” sample, subsequently used for genome sequencing, was determined through flow cytometry of propidium iodide (PI)-stained nuclei, following the procedure described by the CyStain PI Absolute P protocol (Sysmex Partec, Görlitz, Germany). One hundred milligrams of fresh leaf tissue was chopped with a razor blade along with 0.5 ml of Nuclei Extraction Buffer (Sysmex Partec), incubated for 45 min at room temperature and filtered using 30 μm CellTrics (Sysmex Partec). Two milliliters of staining solution (1982 μl of Staining Buffer, 12 μl of PI and 6 μl of RNAse A 3.3 ng/μl) was then added to each filtered sample, and the resulting solution was placed on ice in the dark for 45 min. Analyses were run by setting the following parameters: Nd-YAG green laser: λ = 532 nm; 30 mW, flow rate of 4 μl/s. Raphanus sativus, Glycine max, and Solanum lycopersicum seeds with known 2C DNA content were kindly provided by Prof. Dolezel,1 adopted as reference standards, and their relative fluorescence was used to estimate the genome size of the 2001 sample. Fluorescence histograms were evaluated using FCS Express 5 Flow software (Sysmex Partec), and c-values were inferred by comparing the sample and standard at G0/G1 peak positions.
4
Mesocarp Tissue Ploidy Analysis
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For this analysis, the C-values were measured in a sample of mesocarp tissue derived from a pooling of 5 fruits for each genotype and for each time point for five replicates. Nuclei were isolated from approximately 100 mg of frozen mesocarp by gentle chopping with a razor blade in 0.4 mL of CyStain® PI Absolute P nuclei extraction buffer (Sysmex Partec GmbH, Gorlitz, Germany) supplemented with 1% w/v PVP. Nuclei suspensions were filtered by using 30 μm CellTrics® (Sysmex Partec GmbH, Gorlitz, Germany). Following the filtration step, 1.6 mL staining buffer was added to each sample and tubes were stored in the dark on ice for 1 h before measurement. The fluorescence intensity of DAPI-stained nuclei was determined using the flow cytometer CyFlow® Cube Ploidy Analyzer (Sysmex Partec GmbH, Gorlitz, Germany) equipped with an UV-Light Emitting Diode (l = 355–375 nm). Data were plotted on a logarithmic scale and calibration of C values was made with nuclei suspensions prepared from young leaves. On average, 24,000 nuclei were assessed in each run. Ploidy histograms were quantitatively analyzed with the FCS Express 5 Flow software (Sysmex Partec GmbH, Gorlitz, Germany), after manual adjustment to exclude noise. For each ploidy level, significant differences in the number of nuclei were assessed by Student t tests.
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