Muse cell analyzer assays

SCC-9 and SCC-47 cells were cultured in a 6-well plate with an appropriate number of cells (3 × 10⁵/well) and then exposed to 0, 20, 30, 40 μM platyphyllenone for 24 h. Briefly, the 1 × 105 cells were suspended in 100 μL PBS (contained 2% BSA), and 100 μL of Muse Annexin V & Dead Cell Reagent was added for a total of 200 μL solution in each tube. After incubation in the dark for 20 min at room temperature, Muse Annexin V & Dead Cell Analyzer analysis data was collected using Muse^® Cell Analyzer Assays (Millipore). Each analysis was repeated in three separate experiments.

Cell cycle assay as previously report [27 (link)]. HeLa-Luc or HeLa-shPROK2 cells were collected and fixed with 75% ice ethanol for overnight. Then, these fixed cells were stained with PI reagent for 20 min. Cell DNA content was measured by Muse Cell Analyzer flow cytometry and outcome data further was analyzed by the Muse^® Cell Analyzer Assays (Millipore, Darmstadt, Germany).

The levels of total apoptosis cells were detected by Muse Caspase-3/7 Assay Kit. 1×10⁵ cells treated for 24 h with different concentrations of Polyphyllin G. After PBS washing, prepare cell samples for incubation with Muse™ Caspase-3/7 working solution at 37°C for 30 mim. Add 150 μL of Muse™ MitoPotential 7-AAD and mix thoroughly and run on Muse™ Cell Analyzer and analysis data by the Muse®Cell Analyzer Assays (Millipore).

Huh-7 and Sk-hep-1 cells were treated with 0, 5, 10 and 20 μM of dehydrocrenatidine for 24 h, and subsequently fixed with 75% ethanol. The fixed cells were kept at frozen condition overnight (16–18 h) prior to staining with propidium iodide (PI). The cell cycle distribution was analyzed for 5000 collected cells using Muse^® Cell Analyzer Assays (Millipore, Burlington, MA, USA).

Cells were centrifuged at 800 rpm at 4 °C for 5 min, washed with ice-cold PBS, and stained with propidium iodide (PI) buffer (4 μg/mL PI, 1% Triton X-100, 0.5 mg/mL RNase A in PBS). The DNA content using a Muse Cell Analyzer flow cytometry and analysis data by the Muse^® Cell Analyzer Assays (Millipore, Darmstadt, Germany). Cells were gated to exclude cell debris, doublets, and clumps. The apoptotic cells with hypodiploid DNA content were detected in the sub-G1 region.

The loss of Δψm was detected by JC-1 staining or Muse Mitopotential Assay Kit. For JC-1, after being subjected to indicate treatment, cells painted on the slides and stained with JC-1 staining for 10 min, 37°C. After washing with PBS, the morphological changes related to apoptosis were assessed by fluorescence microscopy (Lecia, Bensheim, Germany). For Muse Mitopotential Assay Kit, 1×10⁵ cells treated for 24 h with different concentrations of Polyphyllin G. After PBS washing, prepare cell samples for incubation with Muse™ MitoPotential working solution at 37°C for 20 mim. Add 5 μL of Muse™ MitoPotential 7-AAD and Incubate at room temperature for 5 min. Mix thoroughly and run on Muse™ Cell Analyzer and analysis data by the Muse®Cell Analyzer Assays (Millipore).

The analysis was conducted by using Muse Mitopotential Assay Kit (Merck Millipore). The SCC-9 and SCC-47 cells were collected at 1 × 10⁵ per tube and treated for 24 h with different concentrations of platyphyllenone (0, 20, 30, 40 μM). After PBS washing, Muse MitoPotential working solution was added to the sample, which was reacted at 37 °C for 20 min. Muse MitoPotential 7-AAD was added soon afterwards, reacting for 5 min at room temperature, and Muse MitoPotential Analyzer and analysis data were obtained using the Muse^® Cell Analyzer Assays (Merck Millipore). Each analysis was repeated in three separate experiments.