Facs lyses buffer

CD19⁺ B cells in fresh whole blood were assessed for ex vivo surface expression of CD27, CD36, TLR2 and TLR4. Neutrophils in fresh whole blood were assessed for TLR2 and TLR4 levels. 100 µl/tube of heparinized whole blood were incubated with fluorescently labeled antibodies (BD Pharmingen; eBioscience) at 4°C for 30 min. Red blood cells were lysed with 2 ml of FACS lyses buffer (BD Pharmingen) for 30 min at room temperature in the dark. Cells were washed with 2 ml of 0.2% bovine serum albumin (BSA) in PBS and evaluated by flow cytometry. Assessment of surface expression on B cells was performed with gates generated with anti-CD19 for each sample. Surface expression on neutrophils was performed using electronically-separated cells on FSC/SSC. The remaining whole blood was centrifuged; the plasma collected and stored at −20°C for the detection of antibodies, inflammatory mediators, and cytokines detailed below.