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. 2023 Nov 8;19(11):e1011585. doi: 10.1371/journal.ppat.1011585

Fig 8. Phagocytosis of PV by primary monocytes in the presence of IgG from malaria-exposed adults.

Fig 8

(A) eFluor 670+ (left panel) and pHrodo+ (right panel) PV isolated from iRBC, as described in Fig 7, were incubated for one hour with primary monocytes in the absence or presence of 80 μg/ml US IgG, and 80 μg/ml Mali IgG. Phagocytosis was determined by quantifying the proportion of eFluor 670+ or pHrodo+ monocytes by flow cytometry. Error bars represent standard error of the mean. Four independent experiments were performed with monocytes from different donors, identified by different colors, and with independent PV preparations. (B) Experiments performed with PV isolated from iRBC (PV) as described in Fig 7, or PV isolated after incubation of iRBC with NK cells (NK-PV) at a NK to iRBC ratio of 6:1 for 3 hours. Due to limitations in the number of PV released and isolated after NK-mediated lysis of iRBC, the no Ab control was performed in 2 out of 6 experiments and the US IgG control in 3 out of 6 experiments. The crucial comparison of US versus Mali IgG was performed in all experiments. Error bars represent standard error of the mean from six independent experiments. (C) Live imaging of primary monocytes stained with eFluor 450 (blue) and co-incubated at a ratio of 1:1 with PKH26-negative eFluor 670+ PV (red) in the presence of 80 μg/ml Mali IgG. Numbers in the top right corner of each image indicate time in minutes. PI was added at the start of image acquisition to exclude damaged monocytes and PV. At time 0, seven PV that were phagocytosed within the next 13 minutes are numbered (yellow) to their left in the order of subsequent phagocytosis by a monocyte. For example, PV1 at time 0, which was phagocytosed after one minute, is labeled 1’. Subsequent phagocytosis events are labeled 2’, 3’, to 7’. The eFluor 670 signal of PV after phagocytosis diminished rapidly, except for PV2, still visible 10 minutes later. PV2 may represent a PV retained in a phagocytic cup prior to complete endocytosis. * p < 0.05, ** p < 0.01 Sidak multiple comparisons test.