Visualizing CpG-Hemozoin Binding in Cells

Five microgram of human CpG-2006 DNA with a phosphorothionate (PTO) backbone bound to Alexa-488 (Integrated DNA Technologies, Coralville, USA) were mixed with 100 µg/ml of sonicated synthetic hemozoin [32] (link)and co-incubated with rocking for 2 h followed by washing of the complex three times with PBS. Bound and unbound DNA were determined by measuring the DNA concentration in the collected supernatant, using the Nanodrop. BL2 and IM 171 cells were each plated at 1 ml (with approximately 500,000 cells per confocal plate). The CpG-Alexa-488-hemozoin complex was added to the cell lines BL2 and SP-IM 171, and incubated for 2 hours after which confocal microscopy was carried out. Confocal reflection microscopy for detection of hemozoin was combined with fluorescence microscopy to detect the Alexa-488 tagged CpG on a Leica SP2 AOBS confocal laser-scanning microscope as described in detail in [54] (link).