Nxt attune facs analyzer

Micronucleus assay was performed using 6–12 weeks of age mice. Mice blood samples were mixed with 100 μl PBS supplemented with 1000 U ml⁻¹ of heparin (Calbiochem). Mixed blood suspension was then added to 1 ml of methanol and stored overnight at −80 °C. Fixed blood cells were washed with bicarbonate buffer (0.9% NaCl, 5.3 mM NaHCO₃). The cells were suspended in 100 μl of bicarbonate buffer with 1 μL of FITC-conjugated CD71 antibody (FITC, eBioscience) at 4 °C for 45 min. The cells were washed with bicarbonate buffer and resuspended in PI/RNase solution (Thermo Fisher) at RT for 30 min. The samples were analyzed an Nxt Attune FACS analyzer (Thermo Fisher) and data analyzed with Flow Jo^{35 (link)}.

Spleen was harvested from mice (6–12 weeks) and ground on 70μm mesh. For quantification of genomic instability, harvested splenocytes were fixed overnight with 70% Ethanol at 4 °C. Fixed cells were permeabilized using 0.25% Triton in PBS with 0.25% Triton-X100 at 4 °C for 10 min. After permeabilization, cells were washed with PBS and stained with γ-H2AX antibody (Millipore) at RT for 2 h. After cells were washed, cells were stained with FITC mouse secondary antibody (Jackson immune Research) at RT for 1 h. After staining, cells were washed and then incubate with PI/RNase solution (Thermo Fisher) at RT for 30 min. The samples were analyzed an Nxt Attune FACS analyzer (Thermo Fisher) and data analyzed with Flow Jo. For metaphase spread, harvested T cells were incubated with concanavalin A (2.5 µg ml⁻¹) for 72 h and colcemid (KaryoMAX, GibcoBRL). After incubation, cells were swollen in prewarmed 75 mM KCl at 37 °C for 20 min. After centrifuge, cells were fixed with carnoy’s buffer (3:1 methanol: acetic acid) at RT for 10 min. The cells were spun down for 4 min at 1000 rpm and then supernatant was aspirated. The cells were resuspended with carnoy’s buffer twice. The cells were dropped on slide and dried for at least 10 min. Slides were stained with Giemsa solution (Sigma)^{34 (link)}. Genomic instability was measured by counting cells that have chromosome breaks and loss.