Acea novoexpress software

After 48 h of exposure to the pre-calculated IC₅₀ value of jatrophone, MCF-7^ADR cells were extracted using trypsin. The cells were then fixed in ice-cold 60% ethanol in PBS at 40 ^oC overnight after being washed twice in phosphate-buffered saline (PBS). Following PBS washing, cells were resuspended in 500 µL of propidium iodide (PI) with RNase staining buffer and incubated for 35 min before readings were obtained from the flow cytometer (ACEA NovocyteTM flow cytometer, ACEA Biosciences Inc. San Diego, California). Data were analyzed using ACEA Novo ExpressTM software (ACEA Biosciences Inc., San Diego, USA) and a minimum of 12,000 cells/events were obtained each sample [30 (link)].

Flow cytometry was used to investigate the influence of quercetin and sorafenib on the cell cycle distribution of HepG2 cells. For 48 h, the cells were treated with either free media (control) or the pre-determined IC50 of quercetin, sorafenib, or a quercetin/sorafenib combination. After incubation, the cells were trypsinized and washed twice with ice-cold PBS before being re-suspended in 0.5 mL of PBS. Two milliliters of 60% ice-cold ethanol were added, and the cells were fixated at 4 °C for 1 h. After being washed, the fixed cells were resuspended in 1 mL of PBS containing 50 g/mL RNase A and 10 g/mL propidium iodide. The cells were examined for DNA content after 20 min of incubation in darkness at 37 °C using an FL2 (ex/em 535/617 nm) signal detector (ACEA NovocyteTM flow cytometer) (ACEA Biosci-ences Inc., San Diego, CA, USA). There were around 12,000 occurrences per sample. ACEA Novo-ExpressTM software was used to calculate cell cycle distribution (ACEA Biosciences Inc., San Diego, CA, USA).

Flow cytometry with two fluorescent channels and an annexin V-FITC apoptosis detection kit were employed to identify apoptosis and necrosis in cell populations (Abcam Inc., Cambridge Science Park, Cambridge, UK) using a published procedure [26 (link)]. After 24/48 or 72 h of treatment with ERF and doxorubicin (10 µM) as a positive control, cells (10⁵) were trypsinized and rinsed twice with ice-cold PBS (pH 7.4). The cells were then maintained at room temperature in the dark for 30 min with Annexin V-FITC/PI solution 0.5 mL, as directed by the manufacturer. After labeling, cells were added to an ACEA Novocyte^TM flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA) and measured for PI and FTIC fluorescent signals with FL1 and FL2 signal detectors (λ_ex/_em 488/530 nm for FITC and λ_ex/_em 535/617 nm for PI, respectively). ACEA NovoExpress^TM software was used to assess the positive FITC or PI cells for each sample, utilizing quadrant analysis (ACEA Biosciences Inc., San Diego, CA, USA).

To assess the effect of isolated alkaloids (solasonine, solasodine and solamargine) on cell cycle distribution, cells were incubated with 5 µM of the test compounds for 24 h and 48 h. Cells were collected by trypsinization; washed twice with ice-cold PBS and re-suspended in 0.5 ml PBS. Two milliliters of 70% ice-cold ethanol was added gently while shaking. Cells were kept in ethanol solution at 4 °C for 1 hour for fixation. Upon analysis, cells were washed and re-suspended in 1 ml of PBS containing 50 μg/mL RNAase A and 10 μg/mL propidium iodide (PI). After 20 minutes incubation in dark place at room temperature, cells were analyzed for DNA contents by ACEA Novocyte™ flowcytometer (ACEA Biosciences Inc., San Diego, CA, USA) and analyzed for PI fluorescent signals using FL2 detector (λ_ex/em 535/617 nm). For each sample, 12,000 events were acquired. Percent of cells in each cell cycle phase was analyzed and calculated using ACEA NovoExpress™ software (ACEA Biosciences Inc., San Diego, CA, USA). Each treatment was repesated three times and data represents mean ± SD of three replicates.

To assess the effect of the selected active compound (paradol) on cell cycle distribution, SAOS-2 cells were treated with 1 µM paradol for 24 h and 48 h and compared to estradiol (0.1 µM). After treatment, cells were collected by trypsinization and washed twice with ice-cold PBS and re-suspended in 0.5 mL of PBS. Two milliliters of 70% ice-cold ethanol were added gently while vortexing. Cells were kept in an ethanol solution at 4 °C for 1 h for fixation. Upon analysis, fixed cells were washed and re-suspended in 1 mL of PBS containing 50 μg/mL RNAase-A and 10 μg/mL propidium iodide (PI). After a 20-min incubation in a dark place at room temperature, cells were analyzed for DNA contents. Cells were injected through ACEA Novocyte™ flow-cytometer (ACEA Biosciences Inc., San Diego, CA, USA) and analyzed for PI fluorescent signals using an FL2 signal detector (λex/em 535/617 nm). For each sample, 12,000 events were acquired, and cell cycle phases were quantified by using ACEA NovoExpress™ software (ACEA Biosciences Inc., San Diego, CA, USA, version 1.1.0) after defining the cell fragment-free fluorescent gate. Ungated events were used to determine cells in the supra-G₂/M phase. The proliferation index was calculated by dividing the total cells in S- and G₂/M-phases by cells in G₀/G₁ phases.

To assess the effect of the TQ, GCB and their combination on cell cycle distribution, MCF-7 and T47D cells were subjected to the pre-determined IC₅₀’s of test drugs or drug free media for 24 and 48 h. After treatment, cells were collected by trypsinization and washed twice with ice-cold PBS and re-suspended in 0.5 mL of PBS. Two milliliters of 60% ice-cold ethanol were added gently while vortexing and cells were incubated at 4 °C for 1 h for fixation. Upon analysis, fixed cells were washed and re-suspended in 1 mL of PBS containing 50 µg/mL RNAase A and 10 µg/mL propidium iodide (PI). After 20 min of incubation in dark at 37 °C, cells were analyzed for DNA contents using flow cytometry analysis FL2 (λ_ex/em 535/617 nm) signal detector (ACEA Novocyte™ flowcytometer, ACEA Biosciences Inc., San Diego, CA, USA). For each sample, 12,000 events were acquired. Cell cycle distribution was calculated using ACEA NovoExpress™ software (ACEA Biosciences Inc., San Diego, CA, USA).