Anticrt alexa fluor 488 conjugate

A2780 cells were seeded on 50 mm glass-bottom culture dishes (Mattek, Ashland, USA) at a density of 2 × 10⁵ cells per dish, and incubated overnight. Then the cells were treated with 1 (1 and 5 μM), doxorubicin (20 μM), oxaliplatin (100 μM) or cisplatin (20 μM) and the treatment continued as described above. Subsequently, the cells were fixed with 4% formaldehyde and stained with antiCRT-alexa fluor 488 conjugate (Abcam). Samples were visualized on a confocal microscope Leica TCS SP8 SMD (Leica microsystems GmbH, Wetzlar, Germany).

A2780 cells were seeded on the 6-well plate at the density of 2 × 10⁵ cells per well and incubated overnight. Then the cells were treated with 1 (1 and 5 μM), doxorubicin (20 μM), oxaliplatin (100 μM) or cisplatin (20 μM), and then further incubated as described above. Subsequently, the cells were fixed with 4% formaldehyde and stained with antiCRT-alexa fluor 488 conjugate (Abcam). Before the analysis on a flow cytometer (BD FACSVerse), the cells were co-stained with propidium iodide. Single cells (3 × 10⁴) were analyzed and plotted on the histogram with a bi-exponential scale. For population analysis, acquired events were plotted on 2D density plots, and the CRTpositive population was identified in CRT+/PI-quadrant; the number of the cells was expressed in percentages from the single gated cells. Data were analyzed using FCS Express 6 (DeNovo software; Glendale, CA).