Endotoxin free pbs

Synthetic Hz was prepared using hemin chloride (Sigma, > 98% HPLC) as previously described (Shio et al., 2009 (link)). The dried pigment was suspended in endotoxin-free PBS (Cellgro, Manassas, VA) and inspected by microscopy for size and other characteristics; the crystalline nature of the crystals was confirmed by reflection confocal microscopy and electron microscopy (Fig. S1b). Pf parasites (3D7 strain) were cultured as previously described (Parroche et al., 2007 (link)). Pf culture stages and parasitemia levels were assessed daily by Giemsa staining and also checked routinely for Mycoplasma. Natural Hz was extracted from the parasite cultures as described in (Parroche et al., 2007 (link)). The dried pigment was suspended in endotoxin-free PBS and stored at 4°C. Synthetic Hz/CpG complexes were prepared by incubating sHz and CpG in a rocker for 2 h and washing the complex three times with PBS. Synthetic Hz/Pf gDNA was prepared by coating sHz with FBS (Atlas Biologicals) overnight and then incubating with Pf gDNA for 2 h followed by three washes with PBS.

Mice were anesthetized with 2.5% (vol/vol) isoflurane and injected i.m. in each calf muscle with a total vol of 50µl per calf. All vaccines consisted of 10 µg protein antigen (OVA-3K, OVA-NP, NP, or mutNP) that was fully adsorbed to 200 µg alum (or combined with other adjuvants, as indicated) and suspended in endotoxin-free PBS (Cellgro). When indicated, additional reagents (BSA, various DNases, trypsin, and/or chymotrypsin) were added as treatments to vaccines immediately before injection. The endotoxin content of each vaccine component was <1 EU/injection, as measured by Limulus Amoebocyte Lysate Assay (Lonza).