Expression of Activation Marker F4/80 on Macrophage Cells Isolated from Mouse Spleen

Protocol Overview

Spleens from individual sham- and MCAO-treated mice were removed and a single-cell suspension was prepared by passing the tissue through a 100 μm nylon mesh (BD Falcon, Bedford, MA). The cells were washed using RPMI 1640 and the red cells lysed using 1× red cell lysis buffer (eBioscience, Inc., San Diego, CA) and incubated for 3 min. The cells were then washed twice with RPMI 1640, counted, and resuspended in stimulation medium (RPMI, containing 10% FBS, 1% sodium pyruvate, 1% L-glutamine, .4% βME. Cardiac blood was collected and placed in heparinized tubes (Fisher Scientific, Pittsburgh, PA). Cells were pelleted and 1× red cell lysis buffer was added to the cell pellet and incubated for 7 min. Cells were washed twice with RPMI, counted and resuspended in RPMI 1640. The brain was divided into the ischemic (right) and nonischemic (left) hemisphere, digested for 60 min with 1 mg/ml Type IV collagenase (Sigma Aldrich, ST. Louis, MO) and DNase I (50 mg/ml, Roche diagnostics, Indianapolis, IN) at 37°C with shaking at 200 rpm. Samples were mixed with a 1 ml pipette every 15 min. The suspension was washed 1× in RPMI, resuspended in 80% Percoll overlaid with 40% Percoll and centrifuged for 30 min at 1600 RPM. The cells were then washed twice with RPMI 1640, counted, and resuspended in stimulation medium. Cells were washed with staining medium (PBS containing 0.1% NaN3 and 1% bovine serum albumin (Sigma, Illinois) and incubated with combinations of the following monoclonal antibodies: f4/80, CD11b (MAC-1), CD74 for 30 mins at 4 degree C. Cells were washed twice and re-suspended in FACS buffer for analysis.