Lumitrace cfda se kit

To analyze the influence of the tested compounds on tumor cell migration, MDA-MB-231 cells attached to the plate’s plastic bottom were separated by a silicone insert from special migration plates (Culture-insert 2 Well 24, ibiTreat); then, the insert was removed, leaving a gap of 500 ± 50 μm (according to the manufacturer’s data) between the cells. The cells were washed twice by PBS to remove cell debris and floating cells and loaded with a fluorescent probe, a (5,6)-carboxyfluorescein succinimidyl ester (CFDA SE) dye (LumiTrace CFDA SE kit, Lumiprobe, Moscow, Russia). The initial solution of CFDA SE at a concentration of 5 mM in DMSO was dissolved in PBS to prepare a 10 μM solution and was added to the cells for 5 min at 37 °C; then, the cells were washed twice with PBS, and fresh culture medium was added. After that, the cells were treated with various concentrations of glycosides and left for 8 and 24 h. Cells treated with culture medium only were used as control. Cell migration into the wound area was then observed under a fluorescence microscope (MIB-2-FL, LOMO, Saint Peterburg, Russia) with an objective ×10 magnification.

The self-made silicon inserts were placed in the center of the wells in a 24-well plate, and MCF-7 cell suspension was added to each well for 24 h. After adhesion, the inserts were removed and the cells were labeled with (5,6)-carboxyfluorescein succinimidyl ester (CFDA SE) dye (LumiTrace CFDA SE kit, Lumiprobe, Moscow, Russia). CFDA SE stock solution at 5 mM in DMSO was dissolved in PBS to prepare a 10 µM solution. The cell culture medium was replaced with this CFDA SE solution for 5 min at 37 °C. Then, the cells were washed twice with PBS and the fluorescent profile of each well with a 25×25 points matrix was scanned at λ_ex = 485 and λ_em = 520 nm using the plate reader PHERAstar FS (BMG Labtech, Offenburg, Germany). The data were processed using MARS Data Analysis v. 3.01R2 (BMG Labtech, Offenburg, Germany).
The investigated compound at a concentration of 1 µM was added to each well (DMSO was used as a control), and the cells were incubated at 37 °C. The scanning of the fluorescent profile of the wells was carried out after 24 h and 96 h of incubation. The data were obtained as a relative fluorescent units and visualized in a 3D graph using SigmaPlot software.

The MCF-7 cells were seeded in a 12-well plate for 24 h. After adhesion, the cells were stained with (5,6)-carboxyfluorescein succinimidyl ester (CFDA SE) dye (LumiTrace CFDA SE kit, Lumiprobe, Moscow, Russia). CFDA SE stock solution at 5 mM in DMSO was dissolved in PBS to prepare a 10 µM solution. The cell culture medium was replaced with this CFDA SE solution for 5 min at 37 °C. Then, the cell layer was washed with PBS twice, the cell culture medium was added to each well, and the compound at a concentration of 10 µM was added to each well for 48 h. DMSO at the same concentration was used as a control.
After incubation, the cells were washed with PBS twice, scrabbed, and collected in 1.5 mL tubes. The intensity of CFDA fluorescence was analyzed with a NovoCyte flow cytometer (Agilent, Austin, TX, USA).