Neuronal CCL2 expression drives inflammatory monocyte infiltration into the brain during acute virus infection

Charles L Howe; Reghann G. LaFrance-Corey; Emma N Goddery; Kanish Mirchia

BILs were isolated as previously described [20] . Briefly, following cardiac perfusion with 50 mL PBS, leukocytes were isolated from Dounce homogenized whole brain tissue using a 30% Percoll gradient centrifuged at 7800 g ave for 30 min at RT in a Beckman F0630 rotor. The floating myelin layer was removed and the leukocytes were collected, strained at 40 µm via gravity, diluted in 50 mL PBS, and centrifuged at 600 g for 5 min at RT in a Beckman SX4250 rotor. The leukocyte pellet was resuspended in 1 mL PBS and underlaid with 1 mL of 1.100 g/mL Percoll to enrich for monocytes and neutrophils. With subsequent centrifugation at 800 g for 20 min at RT in a Beckman SX4250 rotor without brake, the mononuclear leukocytes were collected at the gradient interface, washed in PBS, and resuspended in flow cytometry buffer containing 1% bovine serum albumin and 0.02% sodium azide in PBS.