Guava cell cycle reagent

Cells were synchronized in serum-free MEM for 24 hrs and then cultured in MEM with 10% FBS for an additional 24 hrs. Trypsin treated cells were fixed in 100% ethanol overnight. Fixed cells were stained with Guava Cell Cycle Reagent (Millipore) and cell cycle was analyzed in a Guava Easy Cyte Mini Base System (Millipore) using the supplied statistical software exactly as described previously [16] .

Cells (SKOV-3, MDA-MB-231 and Saos-2) were seeded in 10-cm dishes and were treated with Snol-A at 60–70% confluence. Control and treated cells were harvested by trypsinization and centrifuged at 2000 rpm for 10 min at RT. Then cell pellet was washed with PBS by centrifugation again. 300 µl cold PBS was added to the pellet and mixed with 700 µl of cold 100% ethanol. The cell pellets were kept at −20 °C for 24 h followed by centrifugation twice (450 g at 4 °C for 5 min). Cells were washed two times with cold PBS with spinning down (450 g at 4 °C for 5 min). RNAse (100 μg/ml, Thermo Fisher Scientific) was added and mixed by slow vortex followed by incubation (37 °C, 1–2 h), centrifugation (450 g at 4 °C for 5 min), and cells were re-suspended in 200 µl Guava Cell Cycle Reagent (Millipore) followed by ~30 min incubation in dark at RT. Finally, cells were diluted in 0.5–1 mL volume and samples were acquired using Guava PCA-96 (Millipore) system. Acquired data was analyzed using ModFit LT (Version 5.0) software to distinguish the cell cycle profiles.

Gliomas cells were seeded in a 12-well flat plate at a density of 1 × 10⁴ cells/well (apoptosis assay) or 1 × 10⁵ cells/well (cell cycle assay). After 24 h, KPF-BBR (800 µM NG-97; 1800 µM U251) or KPF-ABR (600 µM NG-97; 1800 µM U251) was added to the respective plates and they were incubated for 72 h.
For the apoptosis analyses, the cells were incubated with 100 μL of Guava Nexin Reagent (Millipore, Billerica, MA, USA), which contained Annexin-V-PE and 7-AAD antibodies.
For the cell cycle analyses, cells were fixed in 1 mL of 70% cold ethanol and incubated at 4 °C for 24 h. After incubation, the cells were centrifuged at 1500 rpm for 5 min and the cell pellets were resuspended in 200 μL of Guava Cell Cycle Reagent (Millipore, Billerica, MA, USA), which contained propidium iodide.
Cell cycle distribution was calculated from 10,000 cells using the Millipore Guava EasyCyte 5HT Benchtop Flow Cytometer (Millipore, Billerica, MA, USA).

To determine the effect of acute exposure to H₂O₂ on the cell cycle by flow cytometry, 500,000 MCF-7 cells were seeded in 25 cm² culture flasks and treated with 25 µM and 250 µM concentrations of H₂O₂ for 24 hours. Similarly equal number of cells chronically exposed to H₂O₂ and their passage matched untreated control were seeded in 25 cm² flask. Semi-confluent actively growing cells with acute and chronic exposure to H₂O₂ were collected and fixed in 70% ethanol for at least 24 hours at 4°C. Just before flow cytometry analysis, fixed cells were collected by centrifugation, washed with ice cold 1X PBS followed by staining with Guava cell cycle reagent (Millipore) and analysed in Guava Easy-Cyte HT Flow Cytometer (Millipore). Cell cycle analysis was carried out by counting 5000 events and acquired data was analysed using Guava Incyte software (Millipore). All samples were analysed in triplicates and experiment was repeated twice.

The cell cycle was analysed as described previously48 (link). The cells were detached with trypsin, collected with the supernatant, and washed twice with ice-cold PBS. The cells were fixed in 75% (v/v) cold ethanol for no less than 12 h and washed in ice-cold PBS. After removing the supernatant, the cells were stained with the Guava^® Cell Cycle Reagent (Millipore). Cell cycle populations were determined using the Guava^® EasyCyte system (Guava Technologies, Hayward, CA, USA), according to the manufacturer’s recommendations. Guava^® Cell Cycle software (Guava Technologies) was used to determine the different cell cycle phases in the cell populations.