Cycletest plus dna reagent kit

The preparation of uniform suspensions of single nuclei was conducted according to the protocol supplied with the Cycle TEST Plus DNA Reagent Kit (Becton Dickinson) with subtle modifications. Specifically, after thawing the sample at room temperature and spinning it down at 12,000 g at 4°C for 5 min, the storage buffer was removed and a volume of 125 µl trypsin buffer was added and mixed by gently tapping the sample tube by hand. The sample was allowed to react with the trypsin buffer for 10 min at room temperature, then 100 µl trypsin inhibitor and RNase-containing buffer was added and the sample tube was again gently tapped by hand to mix and incubated for 10 min at room temperature. Then, 100 µl propidium iodide stain solution was added and incubated for at least 10 min in the dark on ice. Finally, to avoid clogging from cell and tissue fragments, the samples were filtered through 50 µm nylon mesh before flow cytometry analysis. Cellular DNA content was analyzed using an Accuri C6 Flow Cytometer (Becton Dickinson). For each sample replicate, 10,000 nuclei were analyzed using ModFit LT4.1 software (Verity Software House, Topsham, ME, USA) and the cells were classified as being in G0/G1, S and G2/M phase depending on the intensity of the fluorescence peaks (Crissman et al., 1975 (link)).

Cells were seeded in 6‐well plates, and after an overnight recovery period, cells were treated for 24 h with vehicle, 7.5 nm, 12.5 nm, or 20 nm volasertib, corresponding with the IC₂₀, IC₄₀, and IC₆₀ values in A549 cells, respectively. Cell cycle analysis was performed immediately after the treatment period using the CycleTEST™ PLUS DNA Reagent Kit (Becton Dickinson, Erembodegem, Belgium), according to the manufacturer's instructions. Flow cytometric analysis was performed on a FACScan flow cytometer (Becton Dickinson). Each sample was analyzed using 10.000 events/sample acquired. Histograms of DNA content were analyzed using flowjo Software v.10.0.7 to determine the percentage of cells in each phase of the cell cycle.

Cell cycle analysis was carried out on cell cultures grown to subconfluency. Tumor cell populations were stained with PI, using a Cycle TEST PLUS DNA Reagent Kit (Becton Dickinson, city, state if USA, country) and then subjected to flow cytometry using FACScan (Becton Dickinson). A total of 10,000 events were collected from each sample. Data acquisition was carried out using CellQuest software and cell cycle distribution was calculated using the ModFit software (Becton Dickinson). The number of gated cells in G1-, G2/M- or S-phase was expressed as %.

We used fluorescein isothiocyanate (FITC)—labeled annexin V and counterstaining with propidium iodide (Annexin V-FITC Apoptosis Detection Kit, Alexis Biochemicals) according to the manufacturer´s protocol. Cell analysis was carried out with FACS-Calibur (Becton Dickinson, San Jose, CA). In brief, cells were harvested after AMF-treatment for the indicated time periods and 10⁵ cells were transferred into flow cytometry tubes. After washing with 1 ml PBS and centrifugation, 200 μl of annexin V-FITC was added to each tube and incubated for 10 minutes in the dark. Just before analysis, propidium iodide was added to each tube.
For cell cycle analysis cells were collected, washed with PBS, and stained with propidium jodide using CycleTest Plus, DNA Reagent Kit (Becton Dickinson, San Jose, CA) as previously published [3 (link)]. Cell cycle status was analysed on a Becton Dickinson Flow Cytometer (FACS Calibur). Gating strategies using FL3-W channel were applied in order to exclude doublets from the analysis.