Handling and Infection of Bone Marrow Cells

The detailed procedure of handling freshly obtained bone marrows have been described previously15 (link). Briefly, the sample from the original biological container, sometimes a syringe or IV bag, was removed. Care was taken to ensure the sample remained sterile. After initial blood smears to ensure cell viability, cells were treated with RBC lysis buffer (QIAGEN #386516) for 10 minutes on a mild shaker, after ensuring full RBC lysis we centrifuged the cells at 300 g for 6 min. Cells were counted and distributed into tubes before they were infected with Vero-derived dengue virus serotype 2, 16681 strain, at MOI = 0.1 for 2 hours with mild mixing every 20 minutes. Then the cells were washed with serum free RPMI by centrifugation and resuspension three times before they were distributed into different tubes and cultured in 2 ml of 10% FBS RPMI (Gibco #11875–093). Virus was harvested at assigned times.