Muse cell analyzer

To determine the cell number, 1.5 × 10⁵ cells were seeded in 10 cm dishes and allowed to adhere overnight. The following day, cells were treated with drugs as described in Section 2.2. After 9 days of treatment, cells were trypsinized and counted using Muse^TM Cell Analyzer (Merck KGaA, Darmstadt, Germany). For each further experiment where a certain cell count was required, we counted the cells using the Muse^TM Cell Analyzer (Merck KGaA, Darmstadt, Germany).

HUVEC cells were seeded in six-well culture plates at a density of 2 × 10⁵ cells/well. After incubation overnight, HUVEC cells were added to fresh DMEM medium with different concentrations of 8a (0, 3.125, 6.25, and 12.5 μM) for 24 h. After the treatment, cells were harvested into centrifuge tubes and washed three times using precooled PBS buffer. Apoptotic cells were stained with Muse™ Annexin V &Dead Cell assay kit (Muse TM Cell Analyzer, Millipore (catalog no. MCH100105)) according to the manufacturer’s instruction. The cell samples were then analyzed by flow cytometry (Muse TM Cell Analyzer, Millipore, Bedford, MA, USA).

Cell apoptosis was performed by flow cytometry using the Muse^® Cell Analyzer (Merck) and the Muse Annexin V & Dead Cell Assay Kit (Luminex, Austin, TX, USA). Cell suspension is analysed using the MuseTM Cell Analyzer (Millipore Corporation, Burlington, USA).

To quantify cell apoptosis, BXPC-3 cells were treated with different concentrations of compound 8a, Gemcitabine or their combination for a further 72 h. Harvested cells were incubated with Muse™ Annexin V &Dead Cell assay kit (Muse TM Cell Analyzer, Millipore (catalog no. MCH100105)). Cells were then analyzed by flow cytometry (Muse TM Cell Analyzer, Millipore).

Cells were seeded on 6-well plates and incubated with different dose of AA for 24 h. The cells were harvested, washed and suspended in Muse MitoPotential working solution at 37 °C for 20 min. Add 5 μL of 7-AAD and incubate at room temperature for 5 min. Samples were monitored by a Muse Cell Analyzer flow cytometry (EMD Millipore) The data were analyzed by a Muse Cell Analyzer (Millipore).

After the treatment, the cells were washed with phosphate buffered saline (PBS), resuspended in fresh medium, and incubated for 1 h at 37 °C to observe H2AX and ATM phosphorylation, as described previously [45 (link)]. The activation of ATM and H2AX was determined using the Muse^TM Multi-Color DNA Damage kit (Millipore, Hayward, CA, USA) according to the manufacturer's instructions. Following the treatments, the cells were washed with PBS, resuspended in 1× Assay Buffer, mixed with Fixation Buffer (1:1), and allowed to cool. Then, the cells were permeabilized with ice-cold 1× Permeabilization Buffer, resuspended in 1× Assay Buffer, and stained with anti-phospho-ATM (Ser1981)-PE and anti-phospho-Histone H2AX (Ser139)-PECy5 conjugated antibodies for 30 min at room temperature in the dark. The excess of dyes was washed out with ice cold 1× Assay Buffer, and the samples were quantified by Muse Cell Analyzer (Millipore, Hayward, CA, USA). The events for ATM activated cells, H2AX activated cells, cells with DNA double-strand breaks determined as dual activation of both ATM and H2AX, and negative cells, were counted with the Muse Cell Analyzer (Millipore, Hayward, CA, USA) and analyzed with MuseSoft 1.4.0.0 (Millipore, Hayward, CA, USA).