Hoechst 33342 ready flow reagent

After the final wash (see Duolink^® PLA Fluorescence Protocol), 100 µL Ca²⁺/Mg²⁺ PBS was added to the cells. To label the cell bodies, washed cells were incubated with 500 nM SYTO RNASelect Green (Invitrogen, ThermoFisher Scientific, MA, USA, S32703) in Ca²⁺/Mg²⁺ PBS for 20 min at room temperature, and then washed twice with Ca²⁺/Mg²⁺ PBS for 5 min each. To stain the nuclei, one drop of Hoechst 33342 Ready Flow Reagent (Invitrogen, ThermoFisher Scientific, MA, USA, R37165) was added to each dish and incubated for 5 min without washing. After the staining, the cells were imaged in Ca²⁺/Mg²⁺ PBS using a confocal Leica TCS SP5 laser-scanning microscope (Leica, Wetzlar, Germany) equipped with an HCX PLAPO CS 63Ч1.4 oil immersion lens. The image acquisition parameters were as follows: -

Hoechst fluorescence (DNA staining) with excitation at 405 nm and emission at 414–487 nm;

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RNASelect Green fluorescence, excitation at 488 nm, emission at 495–590 nm;

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Duolink Detection Reagent (Red) fluorescence, excitation at 594 nm, emission at 600–652 nm.

Images were processed using LAS X software (Leica, Wetzlar, Germany).

Two drops of Hoechst 33342 Ready Flow Reagent from Invitrogen was added to 2 × 10⁶ cells and incubated for 15 min. Cells were spun, resuspended in Miltenyi Biotec FACs buffer and assayed using the BD FACSAria Fusion 3. Green fluorophores were excited at 488 nM with emission at 530 nM, whereas orange fluorophores were excited at 561 nM with emission at 586 nM. Fluorophores in Phenomex Lightning were detected using the FITC and TRED excitation/emission filters in the Cell Analysis Suite. Hoechst staining was measured at 375 nM with emission at 450 nM. To determine FACs signal overlap between fluorophores and Hoechst, 10,000 cell measurements were read into R, Hoechst signal was split into 100 bins and cell cycle fluorophores scaled from 1 to 100 and intensities were compared.

The HBL-100 and HBL-100/Dox cells were seeded on confocal dishes with glass bottoms and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. The max cell density was 1 × 10⁵ cells/cm². The medium was replaced with fresh medium containing 0.5 or 1.0 μM of the OA compound or 0.5 or 25.0 μM Dox for HBL-100 and HBL-100/Dox cells, respectively. DNA marker Hoechst 33342 Ready Flow Reagent was used as a stock solution (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). Mitochondrial marker LumiTracker Mito TMRE (tetramethylrhodamine, ethyl ester; Lumiprobe, Moscow, Russia) was dissolved in DMSO as a 1.0 mM stock solution. The marker of reactive oxygen species, H₂DCFDA (Lumiprobe, Moscow, Russia), was dissolved in DMSO as a 100.0 μM stock solution. After a 16 h incubation period, the cells were incubated with a serum-free culture medium containing Hoechst 33342 dye (final concentration of 1.0 μM), LumiTracker Mito TMRE dye (final concentration of 1.0 μM) and H₂DCFDA dye (final concentration of 1.0 μM) for 30 min at 37 °C in darkness. Fluorescence at 420 nm (Hoechst 33342), at 510 nm (TMRE) and at 575 nm (H₂DCFDA) of the test cultures (including controls) was analyzed immediately. The cells were observed and photographed under a fluorescence microscope, Olympus CKX 53 (Tokyo, Japan), in darkness.