Pararosaniline

For each treatment condition, 1.0 cm long sections from 20 roots were isolated, washed in ddH₂O and fixed overnight in ethanol:glacial acetic acid (v: v = 3:1) at 4 °C before successively rinsing for 10 min with 100% ethanol, 95% ethanol and 75% ethanol. The treated root sections were stored at −20 °C in 70% ethanol. The root-tips were cut into 1.0–1.5 mm sections and transferred to enzyme mixtures containing 4% cellulose (Sigma), 1% pectolyase (Sigma), and 0.5% pectinase (Sigma) and incubated at 37 °C for 1 h. The root tips were then washed in ddH₂O, hydrolyzed in 5 M HCl for 20 min at room temperature, washed three times in ddH₂O, and stained with Schiff’s reagent (pararosaniline; Sigma-Aldrich) for 1 h at room temperature. To prepare slides for observation of cells, 10 uniform root tips were selected. More than 1,000 cells per slide were randomly selected and photographed using an AxioCam MRc5 and AxioVision v. 4.7 software (Carl Zeiss Microscope, Gottingen, Germany). The average mitotic index was estimated based on 30 images with at least 3000 cells. The mitotic index data were analyzed and plotted using Origin 9.1. $$\mathrm{Mitotic}\mathrm{index}=\frac{\mathrm{Total}\mathrm{number}\mathrm{of}\mathrm{mitotic}\mathrm{cells}\mathrm{per}\mathrm{image}}{\mathrm{Total}\mathrm{number}\mathrm{of}\mathrm{cells}\mathrm{per}\mathrm{image}}$$

Apical root fragments were fixed for 60 min in cold Carnoy’s mixture (absolute ethanol and glacial acetic acid; 3:1, v/v). After washing with ethanol (3 times), root tips were rehydrated, hydrolysed for 1 h in 4M HCl, and Feulgen-DNA-stained using the standard method [46 ] with pararosaniline (applied to selectively stain DNA in a quantitative colorimetric measurement of aldehydes in the Schiff’s test; Sigma-Aldrich, Poznan, Poland). Following 3 times rinsing with SO₂ water (sulphurous acid prepared from sodium metabisulphite and dilute HCl to stop Feulgen staining, to fix the color, and to elute unbound molecules) and after washing with distilled water, root tips were crushed on microscope slides in 45% acetic acid. Squash preparations were frozen on dry ice, dried, and embedded in Canadian balsam. The total number of cells analysed was always 8000 (out of 10 root meristems) at each time point. Selected cells were photographed with E-600 epifluorescence microscope (Nikon). The extinction of Feulgen-stained cell nuclei was measured at 565 nm with a Jenamed 2 microscope (Carl Zeiss, Jena, Germany) and calibrated in arbitrary units (a.u.). A computer system (Forel, Łódź, Poland) was used for image analysis. Approximately 5000 nuclei were collected to evaluate the distribution of the DNA content.

Determination for colony formation was assessed by seeding the Caco-2 (p. 50) in cultures with SPF into six-well plate. The cells were cultured in CGM and the cell density was equal to 100 cells per well. Next, the cells were maintained for 10 days in a CO₂ incubator (5% CO₂ and 95% humidity). The medium was removed and cells were fixed with 4% paraformaldehyde (PFA) and stained with pararosaniline (Sigma Aldrich) for 10 min at room temperature. The CFU were counted as colonies consisting only of 50 or more cells according to the method described previously [24 (link)]. The experiment was performed three times.

In order to perform the wound healing test, the Caco-2 cultures (p. 50) were maintained in 24-well plates, enforcing high confluency. Cell monolayers were manually wounded by scraping cultures with yellow pipette tips (0.5–0.6 mm diameter). After that, cultures were washed twice to remove the detached cells and propagated with SPF in a CO₂ incubator for 24 h. Furthermore, cultures were stained with pararosaniline (Sigma Aldrich, Munich, Germany). Determination of wound closure was performed using AxioVision microscope software. The measurements of distance, obtained between the two edges of a denuded area, were performed five times and repeated independently in triplicate.

Hydroxyurea (HU, 2.5 mM), sodium metavanadate (Van, 200 µM), pararosaniline, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), bovine serum albumin (BSA), polyvinylpyrrolidone (PVP-40), propidium iodide (PI), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. Caffeine (CF, 5 mM) was supplied by Merck, Triton X-100 and pectinase from Aspergillus niger by Fluka, cellulase Onozuka R-10 from Trichoderma viride, and RNase from SERVA. 2-aminopurine (2-AP, 10 mM) and pectolyase Y-23 were obtained from ICN Biomedicals. Other chemicals were obtained from POCH S.A.

Apical fragments of embryo roots and cotyledons were fixed in cold Carnoy’s mixture (glacial acetic acid and absolute ethanol; 1:3; v/v) for 1 h. Following rehydration (70% ethanol, 30% ethanol, distilled water), the roots were hydrolyzed in 4 M HCl for 1 h and stained with Schiff’s reagent (pararosaniline; Sigma-Aldrich, St. Louis, MO, USA) according to the standard methods [83 (link)]. After rinsing in SO₂-water and then in distilled water, fragments of cotyledons from the selected zones and 1.5-mm-long apical segments of the roots were cut off and squashed onto Super-Frost (Menzel-Gläser, Braunschweig, Germany) microscope slides. Following freezing with dry ice, cover slips were removed, and the dehydrated dry slides were embedded in Canada balsam. Nuclear DNA content was evaluated by means of microdensitometry using a Jenamed 2 microscope (Carl Zeiss, Jena, Germany) with the computer-aided Cytophotometer v1.2 (Forel, Lodz, Poland). The Feulgen-stained cell nuclei were measured at 565 nm. Microscopic slides were used also to analyze the mitotic and phase indexes.