Jbw301 mouse monoclonal anti γh2ax antibody

Immunofluorescence staining of γH2AX was performed in absolute 1 × 10⁵ CD34+ cells using a JBW301 mouse monoclonal anti-γH2AX antibody (1:500) (#05-636, Merck) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:500) (#A11001, Thermo Fisher) [26 (link),27 ]. At least 50 nuclei were analyzed in each sample.

NTE were analyzed in CD34+ cells (n = 12 samples from 12 patients) at day 6 after culture for 3 days in untreated medium followed by culture for 3 days in conditioned medium or control medium, respectively. CD34+ cells were grown in the corresponding MSC conditioned medium of the same patient. Immunofluorescence staining of the DNA double-strand-break marker γH2AX [32 (link)] was performed using a JBW301 mouse monoclonal anti-γH2AX antibody (Merck, Darmstadt, Germany) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Thermo Fisher) [72 (link),73 ]. At least 50 nuclei were evaluated in each analysis. Cytogenetic analysis of G-banded chromosomes was performed according to standard procedures [74 ]. At least 25 metaphases were analyzed in each sample according to ISCN 2016 [75 ]. Cell viability was assessed using the CellTiter-Glo luminescent cell viability assay (Promega, Fitchburg, MA, USA) according to the manufacturer’s instructions. Luminescence was measured using a microplate reader (Tecan, Männedorf, Switzerland). ROS were analyzed using the ROS Detection Kit (PromoCell, Heidelberg, Germany) according to the manufacturer’s instructions, which allows the detection of hydroxyl, peroxyl and other reactive oxygen species in live cells. Luminescence was measured using a microplate reader (Tecan).