Dmrb epifluorescence microscope

The MIR503HG knockout hES‐H9 cells were cultured until reaching 80% confluency. Colchicine (ST1173, Beyotime) was added to the medium and incubated for 3 h at 37°C, and then cells were digested into single cell by Accutase (07920, STEMCELL Technologies). The cells were resuspended in 75 mm KCl solution for 30 min and then incubated in a fixative containing ethanol/acetic acid (3:1, v/v) for 20 min at RT. 20 µl cells suspension were applied to each clean slide. Dyed by Giemsa stain (C0131, Beyotime) for 30 min at RT, 20 metaphases were counted for each sample, and then chromosome analysis were performed using the Leica DMRB epifluorescence microscope.

GFP-positive cells were evaluated in whole-mounted tissue, which contains 30% of total scala media area from a cross section image of the cochlea. We counted all the GFP positive configurations in the cochlea form base to apex using the fluorescence microscope. To generate cytocochleograms of GFP-positive cells, tissues were viewed in a Leica DMRB epi-fluorescence microscope with 40x (1.25x digital zoom) objective lens. GFP-positive cell counts were analyzed modifying the KHRI Cytocochleogram Program, Version 3.0. In each field of view, a 0.20 mm scale which was placed in the 10x eyepiece was adjusted along the centers of the pillar cells or flat epithelium. The number of GFP-positive cells for each 0.20 mm segment was calculated for each row. To compare between specimens differing in total cochlear length, each specimen’s data were used to calculate the percent of hair cell loss in 75 intervals (each representing 1.33% of that specimen’s cochlea). Total number of GFP-positive cells in entire whole mounted specimen (apex to base) was assessed and compared; number of cells with each different shape was counted as well. Group differences in total number of GFP-positive cells were evaluated by student T-test and comparisons in IBM SPSS software, version 21.0; p < 0.05 was considered significant.

Confluent cells in 6-well plates were treated with 10 µg/ml N-desacetyl-N-methylocolchicine (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 20 min at 37 °C and then dissociated with 0.025% trypsin. The cell suspension was centrifuged at 1300 rpm for 5 min. After washing with PBS, the cells were resuspended in 1 ml PBS and 3 ml 37.5 mM KCl hypotonic solution for 30 min at 37 °C and then treated with 2 ml methanol/acetic acid (3:1, v/v) at room temperature for 5 min. The cell suspension was centrifuged at 1300 rpm for 5 min and the pellet washed twice with 5 ml methanol/acetic acid for 5 min at room temperature. Two to three drops of cell suspension were dropped onto a microscope slide, which was then air-dried and stained by immersion in fresh Giemsa stain (Sigma-Aldrich, Burlington, USA). The chromosome spread was photographed under a Leica DMRB epifluorescence microscope.

Emerging inflorescences were taken from the six pollen parents used in producing the six mapping populations: three pollen parents with 0B and three with 2B chromosomes. The inflorescences were taken as they emerged from the flag leaf and were fixed in Carnoy’s fixative (6:3:1 ethanol/chloroform/acetic acid) for 24 h. They were then removed and placed in 70% alcohol for a further 24 h, and finally stored in 70% alcohol at 4 °C until analysed.The heads were removed from alcohol and squashes made in acetocarmine stain to find cells at metaphase I. Subsequent analysis of chiasmata used a Leica DM/RB epifluorescence microscope. The frequencies of chiasmata were scored in 100 pollen mother cells (PMCs) in each of the plants. For estimation of chiasma positions, 350 rod bivalents for each family were scored as either distal, interstitial, or proximal after the method of Karp and Jones (1983) (link).

Cells in a confluent T25 flask were treated with 1 μg/ml colchicine (Sigma) for 2.5 h at 37 °C and then dissociated with 0.025% trypsin. The cell suspension was centrifuged at 1300 rpm for 3 min. After washing with PBS, the cells were resuspended in 3 ml PBS and 7 ml 37.5 mM KCl hypotonic solution for 20 min at 37 °C, and then centrifuged at 1300 rpm for 3 min. The pellet was resuspended in 5 ml 3.75 mM KCl solution. After another centrifugation at 1300 rpm for 3 min, the pellet was gently resuspended in 5 ml methanol/acetic acid (3:1, v/v) and incubated for 30 min at room temperature. The cell suspension was then centrifuged at 1300 rpm for 3 min, and the pellet was resuspended in 200 μl methanol/acetic acid. Two to three drops of cell suspension were dropped onto a microscope slide, and the slide was then air-dried and mounted with Vectershield containing DAPI (Vector Laboratories). The chromosome spread was then photographed under a Leica DMRB epifluorescence microscope.