The flow cytometry analysis was performed according to the methods of Marangelli [44 (link)] using the CyFlow Ploidy Analyser (Sysmex-Partec, Goerlitz, Germany) to analyze the leaves of R. roxburghii f. eseiosa polyploidy plants. Young leaf tissue samples of approximately 0.5 cm2 were extracted from each treated seedling, chopped with a blade in 200 μL of extraction buffer (CyStain® UV Precise P, Sysmex-Partec, Goerlitz, Germany), and then filtered through a 30 µm mesh filter (CyStain® UV Precise P, Sysmex-Partec, Goerlitz, Germany) to remove debris. Afterward, 800 μL of DAPI staining solution (CyStain® UV Precise P, Sysmex-Partec, Goerlitz, Germany) was added.
Cystain uv precise p
Manufactured by Sysmex
Sourced in Japan, Germany
The CyStain UV Precise P is a laboratory instrument designed for the analysis and measurement of particles, such as cells, in a sample. It utilizes ultraviolet (UV) light to excite the sample, which then emits fluorescent signals that are detected and analyzed by the instrument. The core function of the CyStain UV Precise P is to provide accurate and reliable data on the characteristics of the particles present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Cyflow ploidy analyser, by Sysmex (3 mentions)
Attune focusing analyzer, by Thermo Fisher Scientific (2 mentions)
Cyflow space flow cytometer, by Sysmex (2 mentions)
Lsm 700, by Zeiss (1 mentions)
Leica sp8, by Leica camera (1 mentions)
Cyflow space, by Sysmex (1 mentions)
Cell lab quanta sc mpl, by Beckman Coulter (1 mentions)
Facsmelody, by BD (1 mentions)
Cyflow ploidy analyzer, by Sysmex (1 mentions)
Nuclei extraction buffer, by Sysmex (1 mentions)
Fcs express 5 flow software, by Sysmex (1 mentions)
Celltrics, by Sysmex (1 mentions)
18 protocols using cystain uv precise p
1
Flow Cytometry for Polyploid Leaf Analysis in Rubus roxburghii
2
Rapid Leaf Nucleus Isolation
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One square centimeter of leaf was cut from the rooting shoots and chopped in 250 μl of nucleus-extraction solution (CyStain UV Precise P, Sysmex, Hyogo, Japan). To purify the nucleus extraction solution, 1 mm2 mesh was used. After purification, 1 ml of staining solution (CyStain UV Precise P, Sysmex, Hyogo, Japan) was added, followed by incubation for 1 min. This solution was applied to an Attune focusing analyzer (ABI, MA, USA), and diploid plants were selected. The diploid plants were planted on solid medium and acclimatized.
3
Flow Cytometry for Genome Size
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Genome sizes and ploidy levels were determined by ow cytometry. For the analysis, 0.5 cm 2 fresh leaf tissue was placed in a petri dish (SPL 10060) and chopped with 500 µL Nuclei Extraction Buffer (CyStain UV Precise P, Sysmex) using a sharp blade. The extraction buffer, containing exposed nuclei, was ltered through a 30 µm-nylon mesh lter into a 3 ml-tube and stained in a staining buffer (CyStain UV Precise P, Sysmex). After a short incubation of 30 s, nuclear suspensions were analyzed using a ow cytometer (Partec PA, Ploidy Analyzer, Germany). Mean DAPI uorescence of the target samples was compared with an internal standard, Oriental-Trumpet "Yelloween" (Ramzan et al., 2016; (link)Kwon et al., 2017) . Genome size (2C) was calculated based on the ratio of relative DAPI uorescence of a sample to that of the internal standard. The calculation procedure was as follows: DNA content of the standard × (mean of relative DAPI uorescence of a sample/mean of relative DAPI uorescence of the standard) (Hembree et al., 2020) (link).
4
Leaf Ploidy Level Determination
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Approximately two or three leaves at the young stage or one or two leaves at the middle and late leaf developmental stages were chopped with a razor blade in nuclei extraction buffer (CyStain ® UV Precise P, Sysmex Partec), and then transferred to the staining buffer (CyStain ® UV Precise P, Sysmex Partec) according to the manufacturer's instructions. The ploidy level of DNA in leaf cells was determined using a CyFlow Ploidy Analyser (Sysmex Partec).
5
Ploidy Determination of Rooting Shoots
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The ploidy of the rooting shoots was determined using flow cytometry. One square centimeter of leaf tissue was cut from the rooting shoots, then chopped and added to 250 µL of nucleus-extraction solution (CyStain UV Precise P, Sysmex Corporation, Kobe, Japan). The solution was filtered through 1 mm2 mesh, after which 1 mL of staining solution (CyStain UV Precise P, Sysmex Corporation) was added, and the solution was incubated for 1 min. The stained solution was then analyzed using a Quantum P (Cytotechs, Ibaraki, Japan) to identify 2n plants. The 2n plants were planted on mineral wool (Grodan, Roermond, Netherlands) and acclimatized in a growth room under 25°C and a 16/8 h light/dark cycle.
6
Flow Cytometry for Genome Size
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Genome sizes and ploidy levels were determined by ow cytometry. For the analysis, 0.5 cm 2 fresh leaf tissue was placed in a petri dish (SPL 10060) and chopped with 500 µL Nuclei Extraction Buffer (CyStain UV Precise P, Sysmex) using a sharp blade. The extraction buffer, containing exposed nuclei, was ltered through a 30 µm-nylon mesh lter into a 3 ml-tube and stained in a staining buffer (CyStain UV Precise P, Sysmex). After a short incubation of 30 s, nuclear suspensions were analyzed using a ow cytometer (Partec PA, Ploidy Analyzer, Germany). Mean DAPI uorescence of the target samples was compared with an internal standard, Oriental-Trumpet "Yelloween" (Ramzan et al., 2016; (link)Kwon et al., 2017) . Genome size (2C) was calculated based on the ratio of relative DAPI uorescence of a sample to that of the internal standard. The calculation procedure was as follows: DNA content of the standard × (mean of relative DAPI uorescence of a sample/mean of relative DAPI uorescence of the standard) (Hembree et al., 2020) (link).
7
Cell Cycle Analysis of Al-Treated Roots
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The Al treatment of the hvatr.g mutant and cv. ‘Sebastian’ was performed as described in the previous section. Cell cycle analysis was performed for control roots and roots treated with 10 μM Al for 7 days. For one experimental replication, 20–30 root meristems were analyzed and three replications per treatment were used. The root tips were mechanically fragmented in a nuclei extraction buffer (CyStain® UV Precise P, 05-5002, Sysmex) and the suspension of nuclei was filtered through a 30-μm nylon mesh in order to remove any debris and stained with a staining buffer (CyStain® UV Precise P, 05-5002, Sysmex). Samples were analyzed with a CyFlow Space flow cytometer (Sysmex, Japan) with a 365-nm UV LED diode as the light source. The flow rate was adjusted to 20–40 nuclei per second. To determine the cell cycle phase, FloMax software with the Cell Cycle Analysis application was used.
8
Ploidy Determination of Rooting Shoots
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The ploidy of the rooting shoots was checked with flow cytometry. One square centimeter of leaf was cut from the rooting shoots, chopped, and added to 250 µl of nucleus-extraction solution (CyStain UV Precise P, Sysmex, Hyogo, Japan). To purify the nucleus-extraction solution, 1 mm2 mesh was used. After purification, 1 ml of staining solution (CyStain UV Precise P, Sysmex, Hyogo, Japan) was added and incubated for 1 min. This solution was applied to an Attune focusing analyzer (ABI, CA, USA), and 2n plants were selected. The 2n plants were then planted on solid medium and acclimatized.
9
Cell Cycle Analysis of Plant Organs
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Cell cycle analysis was performed on lateral roots and leaves. Five root meristems or five leaves were analyzed for one experimental replication and three replications (three plants) per genotype were used. The root tips were mechanically fragmented in a nuclei extraction buffer (CyStain® UV Precise P, 05-5002, Sysmex) and then the suspension of nuclei was filtered through a 30-μm nylon mesh to remove any debris and stained with a staining buffer (CyStain® UV Precise P, 05-5002, Sysmex). The flow rate was adjusted to 20-40 nuclei per second. FloMax software with the Cell Cycle Analysis application were used to determine the cell cycle phase.
10
Visualizing Protein Localization in N. benthamiana
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A. tumefaciens strain AGL1 prepared as above (OD600 = 0.3) and carrying binary vectors was infiltrated into 4-week-old N. benthamiana leaves. Protein localization in epidermal cells of N. benthamiana was assessed 24 or 36 h after infiltration using Leica SP8 (Leica Microsystems) or Zeiss LSM 700 (Carl Zeiss AG) microscopes. For DAPI (4’,6-diamidino-2-phenylindole) staining, the Staining Buffer in CyStain UV precise P (Sysmex America, Inc.) was infiltrated into N. benthamiana leaves using 1 ml needleless syringes 1 h before observation. To image GFP fluorescence, excitation was at 488 nm and emission was collected between 495 and 550 nm. For mCherry fluorescence, excitation was at 555 nm and emission was between 505 and 600 nm. DAPI fluorescence was excited at 405 nm and observed between 410 and 480 nm. Chlorophyll autofluorescence was excited at 633 nm and observed between 638 and 700 nm. Nuclear diameters were measured at their narrowest points (minor axes) using ImageJ 1.51k (Schneider et al., 2012 (link)).
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