Nb100 78356g

Flow cytometry was applied to measure the 8-oxo-deoxyguanosine fraction in nuclear DNA with primary monoclonal antibodies (SC-66036, Santa Gruz, CA, USA) and secondary antibodies (anti-mouse-FITC, SC-2010, Santa Gruz, CA, USA), double-stranded DNA break degree using antibodies to histones H2AX (NB100-78356G, NovusBio, Englewood, CO, USA) conjugated with DyLight488, as well as expression levels of proteins Nrf2 and its phosphorylated form using anti-NRF-2 antibodies conjugated with Alexa Fluor^® 488. Ex: 495 nm, Em: 519 nm (ab194984), and rabbit antibodies to the phosphorylated form NRF-2 (ser40) (bs2013), and secondary anti-rabbit antibodies conjugated with FITC (Sc 2012).

Primary antibodies DyLight488-γH2AX (pSer139) (nb100-78356G NovusBio, Centennial, CO, USA), FITC-NRF2, (bs1074r-fitc, Bioss Antibodies, Inc. Woburn, MA, USA), FITC-BRCA1 (Nb100-598F, NovusBio, Centennial, CO, USA), PE-8-oxo-dG (sc-393871 PE, Santa Cruz Biotechnology, Dallas, TX, USA), CY5.5-NOX4 (bs-1091r-cy5-5, Bioss Antibodies, Inc. Woburn, MA, USA), A350-BCL2 (bs-15533r-a350, Bioss Antibodies, Inc. Woburn, MA, USA), BAX (Nb120-7977, NovusBio, Centennial, CO, USA) and secondary anti-rabbit IgG-FITC (sc-2359, Santa Cruz Biotechnology, Dallas, TX, USA) were used throughout.

MSCs were washed in Versene solution (PanEco, Moscow, Russia) and then treated with 0.25% trypsin, washed with medium, and suspended in PBS. Cells were fixed with paraformaldehyde (PFA, Sigma, 2%, 37°C, 10 min), washed three times with 0.5% BSA-PBS, and permeabilized with 0.1% Triton X-100 in PBS (15 min, 20°C) or with 90% methanol (3 h, 4°C). The cells were washed 3× with 0.5% BSA-PBS and labeled with primary antibodies (1 μg/ml) for 2 h (4°C) and then washed 3× with 0.5% BSA-PBS. The following antibodies were used: γH2AX-Dylight488 (pSer139) (NB100-78356G, Novus Biologicals); NOX4 (Sc-30141, Santa Cruz Biotechnology); 8OHDG (Sc-66036, Santa Cruz Biotechnology); BRCA2 (NBP1-88361, Novus Biologicals); PCNA (ab2426, Abcam); Ki-67FITC (sc-23900 FITC, Santa Cruz Biotechnology); and BCL2 (Sc-783, Santa Cruz Biotechnology). Cells were then incubated for 2 h (20°C) with FITC-conjugated goat anti-rabbit IgG (Sc-2012, Santa Cruz Biotechnology) or goat anti-mouse IgG (Sc-2010, Santa Cruz Biotechnology). To quantify the background fluorescence, we stained portions of the cells with secondary FITC-conjugated antibodies only. To quantify DNA, cells were treated with propidium iodide (PI) and RNase A. The cells were analyzed using a CyFlowSpace flow cytometer (Partec, Germany).