Guava easycyte 6ht 2l benchtop flow cytometer

To examine cell populations in various phases of the cell cycle, the measurement of the DNA content was performed using the Guava Cell Cycle reagent (Merck KGaA) according to the manufacturer's protocol. Briefly, the cells treated with 10 and 20 µM FIS for 24 and 48 h were harvested from 6-well plates, centrifuged (300 × g, 5 min, room temperature) and then fixed in ice-cold 70% ethanol at 4°C followed by overnight incubation at −25°C. The cells were then centrifuged at 650 × g for 5 min at room temperature and washed with PBS. Following centrifugation at 500 × g for 7 min at room temperature, the cells were resuspended in Guava Cell Cycle reagent, and incubated for 30 min at room temperature in the dark. The cells were then analyzed with the Guava easyCyte 6HT-2L Benchtop Flow Cytometer (Merck KGaA), and the percentage of cells in each phase of the cell cycle was determined using InCyte software (version 4.03; De Novo Software).

The cell death in both cell lines was investigated using Guava easyCyte 6HT-2L Benchtop Flow Cytometer (Merck Millipore, Darmstadt, Germany) and double staining with Annexin V Alexa Fluor 488 (AV)/propidium iodide (PI; Thermo Fisher). The cells were cultured in 6-well plates and treated with PL for 24 hours. The next day, after trypsinization and centrifugation, cells were suspended in actin binding buffer (ABB; ThermoFisher). Subsequently, experimental materials were incubated with Annexin V Alexa Fluor 488 for 20 minutes (room temperature [RT], in the dark) and next centrifuged in 300 g ×5 minutes. Then, EA.hy926 and A549 cells were washed with ABB, and stained with PI for 5 minutes (RT, in the dark) and analyzed by cytometer. The data obtained in cell death analysis were examined using FlowJo 10.4.2 software (FlowJo, LCC, Ashland, OR, USA). This assay was also repeated for EA.hy926 and A549 cells after manipulation of PFN1 expression and the treatment with PL.

For the cell death analysis, the AV (annexin V) and PI (propidium iodide) double staining followed by the cytometric measurement using a Guava easyCyte 6HT-2L Benchtop Flow Cytometer (Merck KGaA) were performed. After treatment with 4 µL PL, 1 µM SAN, and 4 µL PL/1 µL SAN, the cells were harvested with 0.25% trypsin (37°C, 5 min; Sigma-Aldrich), centrifugated (8 min x 500g) and suspended in 100 µL of Annexin Binding Buffer (ABB, Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with the addition of 5 µL AV Alexa Fluor 488 (Invitrogen, Thermo Fisher) for 20 min. After centrifugation (5 min x 300g), cells were incubated with 1 µL PI (Invitrogen, Thermo Fisher) for 3 min and analyzed in cytometer and FlowJo vX0.7 software (FlowJo LLC, Ashland, OR, USA). According to the recorded signal, the cells were classified as live (AV-/PI-), early apoptotic (AV+/PI-), late apoptotic (AV+/PI+), and necrotic (AV-/PI+). The studies assessed the type of cell death in A549 cells with the naïve expression of FHOD1 (A549) and A549 cells with the downregulation of the protein (A549 FHOD1-) treated/untreated with the alkaloids.