BrdU Incorporation Quantification in Mice

BrdU was solubilized in 10 mN NaOH / H₂O. 4 mg BrdU (Sigma) in 200 μl PBS was injected intraperitoneally into each mouse 2.5-3 hours before sacrifice^{37 (link),41 ,42 (link)}. Control mice were injected with 200 μl of PBS alone. For quantitation of BrdU incorporation, after isolation and RBC lysis, cell suspensions were fixed in 4% paraformaldehyde (BD) at room temperature for 10 minutes, then washed in 5% FBS followed by PBS. Cells were permeabilized in 100 μl of 0.125% Dawn dishwashing detergent (Procter and Gamble) in 1x PBS for 15 minutes at room temperature, then washed twice with 1% FBS in PBS. Cells were resuspended in DNase buffer (10% FBS in 1 mM CaCl₂ and 0.1 mM MgCl₂) containing 150U of DNase per sample and incubated at 37°C for 45 minutes before washing twice in PBS. Samples were then resuspended in Fc blocking buffer and incubated with antibody cocktail for analysis by flow cytometry.