Anti tlr2 fitc

Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll density gradient and stored with fetal bovine serum (FBS) and 10% dimethylsulfoxide (DMSO) in liquid nitrogen until the time of the assay. Cells were stained with a viability marker (LIVE/DEAD Fixable Violet Dead Cell Stain Kit 405nm, Life Technologies) and different fluorochrome-conjugated antibodies for 35 min at RT to assess the expression of surface marker expression. Anti-CD3, anti-CD19, anti-CD20, and anti-CD56 conjugated with V450 were used as a dump channel. Anti-CD14-BV650 and anti-CD16-PeCF594 (BD biosciences, USA) were used to classify different subsets of monocytes. Anti-TLR2-FITC, anti-HLA-DR-BV570, anti-CD40-APC (all BD Biosciences, USA), and anti-TLR4-BV786 (Biolegend, USA) were determined as activation markers. Anti-CX3CR1-PerCPCy5,5, anti-CCR2-BV605, anti-CD49d- BV711, anti-CD142-PE (all Biolegend, USA), anti-CCR5- APC-Cy7, and anti-CD11b-AF700 (all from BD Biosciences, USA) were used as maturation and homing markers. The cellular markers were analyzed by multiparametric flow cytometry using the Fortessa LSR II cytometer (BD Biosciences, Madrid, Spain). A minimum of 1x10⁶ events were acquired per sample. Data were analyzed using Flowjo 9.3.2 software.

Phenotypic surface staining was performed in BD Pharmingen™ stain buffer (BSA, BD Biosciences, Shanghai, China) for 30 min at room temperature in the dark, using anti-CD14 PE (BD Biosciences, Shanghai, China). Cells were washed and suspended in BD Pharmingen stain buffer (BSA, BD Biosciences, Shanghai, China), anti-TLR-2 FITC, anti-LCK FITC, anti-VAV1 FITC (BD Biosciences, Shanghai, China), was then added separately, and the mixture was incubated for 20 min at room temperature. Finally, cells were washed and acquired on a BD LSRFortessa™ cell analyzer (BD Biosciences, Shanghai, China). The flow cytometry data was deposited in the Flow Repository database [48 (link)] under accession number FR-FCM-Z268.