Cells proliferation was assayed using the Muse Count & Viability Assay (Luminex), according to manufacturer’s instructions. Cells (1 × 105) were seeded in a multiwell-24 plate in 1 mL of complete medium. GEs were added and cells collected 24 h p.t., washed with PBS and resuspended in 50 µL of PBS and 450 µL of Muse Count & Viability Reagent. After an incubation of 5 min at RT, cells were analyzed at the cytofluorometer.
Cell cycle analysis was performed as described [62 (link)]. Cells (2.5 × 105) were seeded in a multiwell-6 plate in 2.5 mL of complete medium. Colcemid (10 µg/mL) was added to synchronize cells in metaphase. After 24 h, colcemid was removed by substituting the culture medium and GEs were administered for additional 24 h. Cells were then collected, centrifuged at 300× g for 5 min and fixated overnight with 200 µL of 70% ethanol. Subsequently, cells were centrifuged and resuspended in propidium iodide (50 µg/mL) and RNAse A (0.1 mg/mL). The samples were analyzed after an overnight incubation at 4 °C by BD FACSLyrics (BD Bioscience, Franklin Lakes, NJ, USA).
Rabbit polyclonal Cyclin E2 (clone H-140) antibody form Santa Cruz Biotechnologies (Dallas, TX, USA) and a mouse monoclonal Cyclin A (clone CY-A1) antibody (Sigma) were used to analyze cell cycles as follows: 2 μg of each antibody were fluorescently conjugated using DyLight555 labelling kit (Biorad, Hercules, CA, USA) as described [63 (link)]. SUP-T1 cells (about 105 for each sample), treated for 24 h with the following micromolar amounts of GEs (i.e., A4 = 70; A5 = 60; B4 = 3.4; B5 = 0.02; C5 = 13.5, according to BIOPEP database) were then fixed and permeabilized using ice cold methanol for 15 min, washed twice with ice cold PBS and then blocked with PBS containing 1% Fetal Calf Serum (FCS) for 30 min at room temperature. After PBS washing, cells were incubated with 2 μL of each fluorescently labelled antibody in a PBS + FCS 1% volume of 50 μL for 2 h at room temperature. Finally, cells were washed twice with D-PBS and analyzed for fluorescence intensity using Guava Muse Cell Analyser (Luminex, Austin, TX, USA), analyzing 2000 events in two independent replicas.
Cell cycle analysis was performed as described [62 (link)]. Cells (2.5 × 105) were seeded in a multiwell-6 plate in 2.5 mL of complete medium. Colcemid (10 µg/mL) was added to synchronize cells in metaphase. After 24 h, colcemid was removed by substituting the culture medium and GEs were administered for additional 24 h. Cells were then collected, centrifuged at 300× g for 5 min and fixated overnight with 200 µL of 70% ethanol. Subsequently, cells were centrifuged and resuspended in propidium iodide (50 µg/mL) and RNAse A (0.1 mg/mL). The samples were analyzed after an overnight incubation at 4 °C by BD FACSLyrics (BD Bioscience, Franklin Lakes, NJ, USA).
Rabbit polyclonal Cyclin E2 (clone H-140) antibody form Santa Cruz Biotechnologies (Dallas, TX, USA) and a mouse monoclonal Cyclin A (clone CY-A1) antibody (Sigma) were used to analyze cell cycles as follows: 2 μg of each antibody were fluorescently conjugated using DyLight555 labelling kit (Biorad, Hercules, CA, USA) as described [63 (link)]. SUP-T1 cells (about 105 for each sample), treated for 24 h with the following micromolar amounts of GEs (i.e., A4 = 70; A5 = 60; B4 = 3.4; B5 = 0.02; C5 = 13.5, according to BIOPEP database) were then fixed and permeabilized using ice cold methanol for 15 min, washed twice with ice cold PBS and then blocked with PBS containing 1% Fetal Calf Serum (FCS) for 30 min at room temperature. After PBS washing, cells were incubated with 2 μL of each fluorescently labelled antibody in a PBS + FCS 1% volume of 50 μL for 2 h at room temperature. Finally, cells were washed twice with D-PBS and analyzed for fluorescence intensity using Guava Muse Cell Analyser (Luminex, Austin, TX, USA), analyzing 2000 events in two independent replicas.
Free full text: Click here
