Surface marker antibodies

Mice were euthanized by CO₂ asphyxiation. Spleen was collected and placed into a wet 40 μm cell strainer on top of a 50 mL centrifuge tube. Then the spleen was smashed in the cell strainer, which was rinsed with 5 mL of medium. The cell suspension collected in the tube was centrifuged at 1,000 rpm for 5 minutes. The pellet was re-suspended in 5 mL red blood cell lysis buffer (containing 0.15 M NH4Cl, 0.01 M KHCO3, and 0.1 Mm EDTA), and incubated at 4°C for 5 minutes. Then the cell suspension was centrifuged and rinsed with PBS (containing 5% FBS). Splenocytes in the final pellet were suspended in RPMI1640 medium containing 20% FBS for further applications. A portion of the splenocytes was stained with surface-marker antibodies (BD Biosciences, Franklin Lakes, NJ, USA) for macrophages. The gating strategies were CD45⁺CD11b⁺Gr-1^-F4/80⁺. The samples were analyzed on an LSRII flow cytometer (BD Biosciences), and FlowJo (Tree Star, Ashland, OR, USA) was used to analyze the results. The data were presented as % positive cells.