Lsm 900 airyscan 2 confocal system

Three-dimensional (3D) cell images were acquired for detailed analyses of cancer cells having bipolar, chromosome ring, or multipolar mitoses. This was done after vehicle or CDK2 inhibitor treatments by use of Z-stack image analyses (Range:11 μM, slices: 12 images, Interval: 1 μM) using the Zeiss Axiocam 506 color microscope with the LSM 900 Airyscan 2 Confocal system (Carl Zeiss Microscopy LLC, White Plains, NY).

Studied cancer cells were fixed in ice-cold methanol (176845000; Thermo Fisher Scientific, Waltham, MA) and stained with Centrin 1 and γ-tubulin or α-tubulin antibody along with Hoechst staining. Cells were mounted with Pro-Long Gold antifade reagent (P36934; Invitrogen) and scored for chromosome rings and for the presence of multipolar anaphases using a ZEISS Axiocam 506 color microscope with
the LSM 900 Airyscan 2 Confocal system (Carl Zeiss Microscopy LLC, White Plains, NY). Primary antibodies recognized: Centrin 1 (12794–1-AP, 1:1000, Proteintech, Rosemont, IL), α-tubulin (T6199; 1:1000, Sigma-Aldrich, St. Louis, MO) or γ-tubulin (T5326; 1:1000, Sigma-Aldrich, St. Louis, MO), respectively. Secondary antibodies were Texas red anti-murine IgG (H+L) (TI-2000; 1:1000, Vector Laboratories, Newark, CA) and goat anti-rabbit IgG (H+L) Cross-Adsorbed Alexa Fluor™ 488 (A11008;1:1000, Thermo Fisher Scientific, Waltham, MA), respectively. Hoechst 33,342 (62249, Thermo Fisher Scientific, 1:10000) stained for DNA. Pro-Long Gold anti-fade reagent preserved immunofluorescence. Each assay was performed in triplicate. Independent triplicate biologic replicate experiments were done.