Metaphase Spread Analysis of Splenocytes

For metaphase spreads of splenocytes^{70 (link)}, freshly harvested spleens were minced and filtered through a 40 μm cell strainer into warm PBS. Cells were spun at 1000 × g for 5 min, resuspended in warm DMEM supplemented with 10% FBS, 100 U/mL penicillin,100 U/mL streptomycin. Cells were then treated with colcemid (KaryoMAX, GIBCO) at 0.1 μg/ml for 1 h at 37 °C^{74 (link)}. Cells were swollen in prewarmed 75 mM KCl for 30 min at 37 °C, then carefully fixed in methanol: acetic acid (3:1) and kept at −20 °C. Metaphase spreads were prepared by dropping cells in the fixative onto Superfrost glass slides (Fisher Scientific) at 25 °C and 60% of humidity. After air dry, metaphase images were captured and analyzed using a 20X Nikon objective (PL APO, 1.4 NA). At least 10 metaphases from each tissue were scored for chromosomal aberration.