Nycodenz gradient

Freshly isolated primary MLiECs were obtained from the liver of the control and DHX15^+/− mice. MLiECs were purified after collagenase A retrograde perfusion (Roche Diagnostics, Basel, Switzerland) and Nycodenz gradient (Sigma-Aldrich, Darmstadt, Germany). The purification was optimized using magnetic beads CD146⁺ (MACS system, Miltenyi Biotec, Bergisch-Gladbach, Germany). Cells were cultured in Microvascular Endothelial Cell Growth Complete Medium (Pelobiotech, Planegg, Germany) in humified atmosphere at 37 °C and 5% CO₂.

BALB/c mice were infected by intraperitoneal injections with Plasmodium yoelii 17XNL expressing GFP, obtained from the Malaria Research and Reference Reagent Resource Center (MR4). Highly purified samples of trophozoite and schizont stage parasites were obtained by Nycodenz gradient (Sigma) as previously described29 (link). Parasites were divided into two equal aliquots of ~100 μl each. The two aliquots were lysed for 4 min at room temperature in presence of protease inhibitor (Pierce) either with 1600 units of Streptolysin O (Abcam) or with 0.05% of Saponin (Sigma) and separated into pellets and supernatants by centrifugation. Prior to resuspension in SDS sample buffer, the pellet fractions were washed twice using RPMI whereas the supernatant fractions were solubilized immediately. The whole procedure was repeated until four biological replicates were obtained.