Anti cd68 pe cy7

To distinguish between PBEC and MQ in PBEC-ALI/MQ, cells were trypsinized and washed twice with PBS, and incubated with monoclonal antibody (anti-CD68-PE-Cy7; Cat No.25-0689-42, eBioscience, Thermo Fisher Scientific, Pittsburgh, United States). The CD68 ⁺ cells were MQ and CD68 ⁻ cells were PBEC. To analyze the expression of TLR2 and TLR4, the trypsinized cells were incubated with monoclonal antibodies (anti-TLR2-APC, Cat No.558319, BD Pharmingen, San Diego, CA, United States; anti-TLR4-PE, Cat No.12-9917-42, eBioscience, Thermo Fisher Scientific, Pittsburgh, United States) for 30 mins in dark. The cells were then washed 3 times and re-suspended in PBS. For PBEC-ALI, the TLR2/TLR4 were detected directly. For PBEC-ALI/MQ, the cells were gated by anti-CD68 antibody first followed by TLR2/TLR4 detection in both CD68 ⁺ cell populations (MQ) and CD68 ⁻ cell populations (PBEC). For all analysis, unstained cells were used to provide the gating controls for determining positivity [46 (link)]. Analyses were performed using the flow cytometer (LSR Fortessa™, BD Bioscienc, United States) and calculated as median fluorescence intensity (MFI).