Anti cd68 fa 11

To analyze splenic lymphocyte populations, organs were removed from mice and RBCs lysed using ACK lysis buffer. Fc block was added prior to antibody staining. The following mAbs were used: anti-TCRβ (H57597), anti-CD122 (TM-beta1), anti-NK1.1 (clone), anti-MSR1 (REA148), anti-CD36 (HM36) and anti-CD68 (FA-11) were purchased from eBiosciences. Isotype controls were purchased from BD Biosciences. BODIPY 495/503 was purchased from Life Technologies (D3922). The following human antibodies were used: anti-CD3 (SK7) anti-CD56 (NCAM), CD36 (CB38), granzyme B (GB11) and perforin (δG9), from BD Biosciences. Flow cytometry acquisitions were performed on a CytoFLEX 30 Summit instrument (Beckman Coulter). Data was analyzed with CytExpert software. Bioimaging was performed using a fluorescence microscope (Leica).

For FCM analysis of cell surface markers, cells were stained with antibodies in PBS containing 0.1% (wt/vol) BSA and 0.1% NaN₃, as described previously^{22 (link)}. Anti-CD11b (M1/70), anti-F4/80 (BM8), anti-CD14 (61D3), and anti-CD68 (FA11) were obtained from eBioscience. Anti-CD45 (TU116) was obtained from BD Biosciences. Anti-CD3 (145-2C11) and anti-CD19 (6D5) were obtained from Miltenyi Biotec. For intracellular staining, TNFα expression (MP6-XT22; Biolegend) was analyzed by flow cytometry according to the manufacturer’s instructions. For TNFα expression analysis, cells isolated from the indicated organs were restimulated with LPS (L2630; Sigma-Aldrich, St. Louis, MO, USA) for 5 h for the CD11b⁺ F4/80⁺ macrophage analysis, while GolgiStop (554724; BD Biosciences) was added for the last 2 h of incubation. After surface staining and washing, the cells were fixed immediately with Cytofix/Cytoperm solution (554714; BD Biosciences) and were stained with anti-TNF-α antibody (MP6-XT22) purchased from eBioscience. Flow cytometry data were acquired on a FACSCalibur instrument (Becton Dickinson, CA, USA), and data were analyzed with FlowJo (Tree Star, San Carlos, CA, USA).