Eryptosis of iRBC after co-culture with PBMCs or purified NK cells.

<p>iRBC were co-cultured 3∶1 for 24 hours with NK cells, NK cells+TKD (5 day stimulation), PBMC, PBMC+TKD (5 day stimulation) of the same donor. 0.5×10⁶ erythrocytes were washed in 5 mM Ringer solution. Afterwards, cells were stained for 15 minutes with Annexin-V (1∶500) and propidium iodide (1∶50). Eryptotic cells were determined as Annexin-V-positive (AV⁺) and propidium iodide-negative (PI⁻). A: Baseline levels of eryptotic uRBC and iRBC at the start of the experiment and after 24 h of culture in growth medium without leukocytes. RBC were stained with AV+PI or left unstained. Gating was done based on FSC/SSC properties and the unstained control. AV was measured in the FL1 channel and PI in the FL2 channel. B: Percentage of AV⁺/PI⁻-iRBC after co-culture in growth medium (GM) or with different effector cells in relation to starting parasitemia (** p≤0.01, student's <i>t</i> test, n = 6). C: Normalized FSC of iRBC after co-culture in growth medium (GM) or with different effector cells (* p≤0.05, student's <i>t</i> test, n = 6). FSC values of co-cultured iRBC were normalized to untreated iRBC (GM). D: PI⁺ necrotic iRBC after culture in growth medium (GM) or with various effector cells. Numbers of necrotic cells were normalized to untreated iRBC (GM).