Fc receptor blocking

Blood was drawn from WD-fed mice in the presence of anti-coagulant (EDTA-2K). After Fc receptor blocking (#553141, BD), cells were stained with fluorescently labelled antibodies F4/80-Alexa488 (#MF48020, 1:20, invitrogen), CD11b-PE (#561689, 1:20, BD) and CD45-PerCP (#561047, 1:20, BD Biosciences) followed by FACS Lysing Solution (#349202, BD Biosciences), according to the instruction manual. Circulating foam cell cluster in high SSC subsets of F4/80⁺CD11b⁺CD45⁺ were measured by Attune Acoustic Focusing Cytometer (Applied biosystems) with modifications as reported previously^{56 (link)}.

After isolations, BM-MNCs were resuspended in FC receptor blocking (BD Biosciences) and incubated on ice for 30 min. Cells were co-stained for 30 min on ice in the dark with Anti-CD133 Magnetic Microbeads (Miltenyi Biotec). Wash cells with PBS and centrifuge at 400 × g for 10 min. Aspirate supernatant and resuspended in PB buffer. Prebalance the LS column (Miltenyi Biotec) with 3 ml PBS, apply cell suspension onto the column that has been fixed in the magnetic Separator (Miltenyi Biotec). Wash column with 10 ml PBS. Remove column from separator and flush out the magnetically labeled cells into 15 ml collection tube with 5 ml PBS. Centrifuge the magnetically labeled cells at 400 × g for 10 min. Subsequently, the pellet containing CD 133 + cells was resuspended in appropriate FACS buffer for further sorting.

The AuNPs exposed BMDMs and BMDCs were stimulated with 2 µg/mL LPS from E. coli, for 24 h at 37 °C with 5% CO₂. The supernatant was harvested for cytokine immunoassay, and the cells were labelled with antibodies specific for CD11b (Ozyme, Saint Cyr, France, cat. no.: BLE101226) and CD11c (Ozyme, cat. no.: BLE117318), or CD11b (Ozyme, cat. no.: BLE101216) and F4/80 (Ozyme, cat. no.: BLE123152), cell surface markers of BMDCs and BMDMs, respectively, after Fc receptor blocking (BD Pharmingen, Claix, France, cat. no.: 553142) to reduce non-specific binding. To evaluate cellular activation, BMDCs and BMDMs were immunostained with anti-IAb-A^b (Ozyme, cat. no.: BLE116410) and anti-CD86 (Ozyme, cat. no.: BLE105008) antibodies. In both cases, only live cells were selected by staining with 7-Aminoactinomycin D (7AAD) (negative gating) (BD Biosciences, cat. no.: 559925) and analysed by flow cytometry using BD™ LSR II (BD Biosciences). The proportion of activated cells was quantified using FCS Express V6 (De Novo Software).