Cy5 labeled annexin 5

Spodoptera frugiperda (Sf) 9 cells (American Type Culture Collection CRL-1711) were cultured in Sf-900Ⅲ SFM medium (Gibco) and TMEM16F^−/−Xkr8^−/− Ba/F3 cells (16 ) in RPMI1640 containing 10% fetal calf serum and 45 units/ml of IL-3.
An hBSG mAb (clone: XBA18) and its Fab fragment were previously described (17 (link)). Cy5-labeled Annexin V was from BioVision. LMNG and fluorinated Fos-choline 8 were from Anatrace. Soybean Polar Lipid Extract was from Avanti. Cholesteryl hemisuccinate (CHS) and tobacco etch virus (TEV) protease fused to a histidine tag were from Sigma–Aldrich. N-dodecyl-β-d-maltopyranoside was from Dojindo. Methyl-β-cyclodextrin (MβCD) was from Tokyo Chemical Industry Co. A recombinant MSP1E3D1 protein was produced in Escherichia coli using its expression plasmid (Addgene) and was purified essentially according to a previously reported protocol (34 ).

Zymosan-induced peritonitis was performed essentially as described (Cash et al., 2009 (link)). In brief, female mice (n = 5–10 per group) at 8–12 weeks of age were injected i.p. with zymosan A particles (250 mg/kg, 0.5 ml saline). Peritoneal lavage fluid was harvested with 5 ml PBS containing 1% FCS at 2 or 6 hr after the injection. Cells and residual zymosan particles were removed by centrifugation at 900×g for 4 min. Mice were grouped randomly, and studied without being blinded.
To detect apoptotic cells, peritoneal cells were suspended in PBS containing 2% FCS, incubated on ice with 1 mg/ml of biotinylated anti-Ly6B.2 (AbD Serotec, UK), followed by the incubation with 2 μg/ml PE-Cy7-labeled streptavidin (BD Pharmingen) and 5 μg/ml FITC-labeled anti-Ly6G (1A8; BD Pharmingen). After washing with Annexin V binding buffer, cells were stained with 2000-fold diluted Cy5-labeled AnnexinV (Biovision, Milpitas, CA) and 500 nM Sytox blue (Invitrogen) and analyzed by flow cytometry.