Cd19 af488

PBMCs (1 × 10⁶) were stained with fixable blue dead cell stain (ThermoFisher, Waltham, Massachusetts and USA) or Zombie NIR™ Fixable Viability Kit (Biolegend, London, UK). This was followed by washes and surface marker staining with antibodies CD4-BUV395, CD8-AF700, CD19-AF488, and CD14-BV711 (Biolegend, London, UK) followed by subsequent washes. Following surface staining, to determine the expression of membrane lipid rafts cells were subsequently stained with a surrogate lipid raft marker CTB¹⁶, conjugated to FITC (1:100 dilution in PBS) (Sigma, Dorset, UK) followed by subsequent washes and fixation in 2% PFA before running on the flow cytometer. Altneratively, cells were fixed filipin complex (Sigma) as described previously [74 (link)]. Stains and washes were carried out in cell staining buffer (Biolegend). Appropriate unstained controls were used for lipid stains.
Data was acquired using a LSRFORTESSA X-20 (BD, Wiltshire, UK) flow cytometer and analysis performed using FlowJo Single Cell Analysis Software (TreeStar). Application settings were created and Cytometer Setup and Tracking (CS&T) (BD) beads were run to assess cytometer performance for each experiment.