Splenic Cell Oxidative Burst Assay

Splenic cells were isolated as described previously and approximately 1 million cells were blocked with 1 µl of TruStain FcX (anti-mouse 16/32) antibody (Biolegend, San Diego, CA) for 1 h on ice. After blocking, 1 µl of different antibodies including anti-CD11b, anti-Ly6G, and anti-Gr1 (All BioLegend, San Diego, CA) were added to each tube together with 1 µl of live/dead cell stain (Invitrogen, Waltham, MA). Hanks’ Balanced Salt solution was used for immunostaining, washing, and resuspending cells. Then 4 µl of dihydrorhodamine 123 (DHR; 5 mM) and 10 µl of N-formyl-Met-Leu-Phe (fMLP) bacterial peptide (10 mM) were added to cells, resuspended in 500 µl of Hanks’ Balanced Salt Solution (HBSS), incubated at 37°C for 20 min and then immediately transferred to ice for 10 min to stop the reaction (37 (link)). Cells were washed twice with HBSS, and fluorescence intensity was measured by flow cytometry.