Metaphase Spread Preparation from MEFs and Splenocytes

To prepare metaphase spreads from MEFs, 1–2 × 10⁶ cells (at passage 5) were treated with 0.05 μg/ml colcemid (GIBCO BRL) for 4–5 h at 37°C. To prepare metaphase spreads from splenocytes (Rudolph et al., 1999 (link)), spleens were freshly collected and minced between two microscope slides. Released cells were suspended in 5 ml PBS, centrifuged at 1,000 rpm for 5 min, resuspended in 4 ml of RPMI containing 10% FBS, IL-2 (10 U/ml), PHA (5 μg/ml), conA (5 μg/ml), and colcemid (0.05 μg/ml), and cultured for 6–7 h at 37°C. After colcemid treatment, MEF or splenocyte cells were harvested, suspended in 5 ml 0.075 M KCl, and incubated at RT for 30 min. Cells were fixed in Carnoy's solution (75% methanol, 25% acetic acid), washed, and finally resuspended in ∼0.5 ml fixative. 25-μl aliquots were dropped onto prewetted microscope slides, stained for 10 min in 5% Giemsa solution, and analyzed on an Olympus AX70 microscope using a 100× objective.