Luciferase Assay for PfEMP1-Mediated iRBC Stimulation

RAW-ELAM (35 (link)) cells (1 × 10⁵ cells/well) in phenol red-free DMEM were added to white 96-well plates and incubated for 12 to 16 h. Medium was removed, and purified iRBCs or uRBCs (5 × 10⁵ cells/well) in 100 μl medium were added. Plates were incubated for a further 12 h. BriteLite Plus luciferase reagent (100 μl; PerkinElmer) was added to wells, and the wells were incubated for 3 min. Total luminescence was measured on a Chameleon V plate reader (Hidex). Samples were analyzed in quadruplicate, and the vehicle negative control and the Ultrapure LPS-EB (lipopolysaccharide from E. coli 0111:B4; InvivoGen) positive control were included. Values for uRBC samples were subtracted from those for iRBC samples. Data were normalized to represent fold changes of PfEMP1-null iRBC stimulation over WT iRBC stimulation and of LPS stimulation over PBS stimulation, where the value for the WT was equal to “1,” and the luciferase levels measured from PfEMP1-null cells were determined relative to the WT signal. Independent experiments were repeated a minimum of three times on separate days and with freshly isolated iRBCs.