Facsaria 2 sorp flow cytometer and sorter

Suspensions of intact mitotic metaphase chromosomes from the line WT153397 were prepared from synchronized root tips cells of young seedlings following Vrána et al. (2016a (link); 2016b (link)). Prior to flow cytometric analysis, chromosomes in suspension were fluorescently labeled by FISHIS (Giorgi et al. 2013 (link)) with oligonucleotide probe 5′-FITC-(GAA)₇-FITC-3′ (Integrated DNA Technologies, USA) and stained with DAPI. Bivariate flow karyotyping and chromosome sorting were carried out using a FACSAria II SORP flow cytometer and sorter (Becton Dickinson Immunocytometry Systems, San José, USA) as described by Molnár et al. (2016 (link)) and Said et al. (2019 , 2022 (link)). The chromosome samples were analyzed at a rate of 900–1400 particles per second, and bivariate flow karyotypes of FITC vs. DAPI fluorescence were acquired. The sorting window was set to the chromosome population of interest on the dot plots, and chromosomes were flow-sorted at a rate of 15–24 per second. Approximately 3000 chromosomes from each chromosome fraction were flow-sorted onto a microscope slide into a 3 µL drop of PRINS buffer supplemented with 2.5% sucrose (Kubaláková et al. 1997 (link)). The chromosome content of the flow-sorted fractions was determined using FISH, as described by Kruppa et al. (2016 (link)).

High molecular weight (HMW) DNA was prepared according to Šimková et al. (2003) (link) with modifications. Four batches of 700,000 G1-phase nuclei each were sorted into 660 μl IB buffer in 1.5 ml polystyrene tubes using a FACSAria II SORP flow cytometer and sorter (BD Biosciences, San Jose, CA, United States). One 20 μL agarose miniplug was prepared from each batch of nuclei. The miniplugs were treated by proteinase K (Roche, Basel, Switzerland), washed in wash buffer (10 mM Tris, 50 mM EDTA, pH 8.0) four times, and subsequently five times in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). After the plugs had been melted for 5 min at 70°C and solubilized with GELase (Epicentre, Madison, WI, United States) for 45 min, DNA was purified by drop dialysis against TE buffer (Merck Millipore, Billerica, MA, United States) for 90 min.

Liquid suspensions of mitotic chromosomes were prepared from root tips of 4AL‐TM seedlings as described by (Vrána et al., 2000). The telosomes were separated from the rest of the genome by flow sorting, using a FACSAria II SORP flow cytometer and sorter (BD Biosciences, San Jose, CA). The level of contamination within a sorted peak was determined using FISH, based on probes detecting telomeric repeats, the Afa repeat and (GAA)_n, following the methods described by Kubaláková et al. (2003). The flow‐sorted 4AL‐TM telosomes were treated with proteinase, after which DNA was extracted using a Millipore Microcon YM‐100 column (www.millipore.com). Chromosomal DNA was MDA amplified using the Illustra GenomiPhi V2 DNA amplification kit (GE Healthcare) as described by Šimková et al. (2008).

Barley metaphase chromosomes (Hordeum vulgare L. cv. Morex) were sorted according to Lysák et al. (1999 (link)). Briefly, a chromosome suspension was prepared from synchronized primary roots meristems. Chromosomes were DAPI-stained, immediately analyzed, and flow-sorted using a FACSAria II SORP flow cytometer and sorter (BD Bioscience, San Jose, CA, USA). Five thousand chromosomes were sorted into 15 μl of PRINS buffer supplemented with 2.5% sucrose (10 mM TRIS, 50 mM KCl, 2 mM MgCl₂.6H₂O, 2.5% sucrose; pH 8) onto high precision coverslips (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany). Before immunolabeling, the coverslips were stored at −20 °C.