Quinacrine mustard

Conventional cytogenetics techniques were performed as described in [7 (link)] in order to determine the effect of 48 h of treatment with 500 nM Danusertib on the mitotic index and cell ploidy. Briefly, cells were treated with 0,2 μg/ml of Colcemid (Roche) and then harvested and incubated with a hypotonic solution of 0,56% w/v of KCl for 15 minutes at room temperature. Subsequently, cells were fixed with a fixative solution composed of 3:1 methanol:acetic acid. Chromosomes were QFQ banded using quinacrine mustard (Roche) and slides were mounted in McIlvaine buffer. Slides were analysed using Nikon Eclipse 80i fluorescence microscope (Nikon) equipped with a COHU High Performance CCD camera. The mitotic index was evaluated counting the percentage of mitosis scoring at least 1000 nuclei, while ploidy was investigated by evaluating the number of chromosomes/metaphase on 30/50 metaphases. Chromosomes spreads were analysed using the Genikon software. Data were obtained as mean values derived from two independent experiments.

The mitotic index was assessed in order to evaluate the effect of ibrutinib on cell proliferation. 2 × 10⁶ cells were seeded in T-25 cm³ in 5 ml of medium. Subsequently, cells in exponential growth phase were treated with 20 μM ibrutinib for 48 h. Then metaphase chromosome spreads were obtained using standard procedures (Riva et al., 2014 (link); Cilibrasi et al., 2017 (link)).
The chromosomes were QFQ-banded using quinacrine mustard (Roche) and slides were mounted in Mc Ilvaine buffer. Slides were analyzed using Nikon Eclipse 80i fluorescence microscope (Nikon) equipped with a COHU High Performance CCD camera. Mitotic index was evaluated counting the percentage of mitosis, scoring at least 1,000 nuclei. Data were obtained as mean values derived from two independent experiments.

The Mitotic Index (MI) was assessed in order to evaluate RSV effect on cell proliferation. 2x10⁶ cells were seeded in T-25 cm³ in 5 ml of medium. Subsequently, cells in exponential growth phase were treated with 100 μM RSV for 48 hs. Then metaphase chromosome spreads were obtained using standard procedures as previously described [28 (link)].
The chromosomes were QFQ-banded using quinacrine mustard (Roche) and slides were mounted in McIlvaine buffer. Slides were analyzed using Nikon Eclipse 80i fluorescence microscope (Nikon) equipped with a COHU High Performance CCD camera. MI was evaluated counting the percentage of mitosis scoring at last 1000 nuclei. Data were obtained as mean values derived from two independent experiments.