Apc antihuman cd45 clone hi30

Leukocytes were transferred to 96-well U-bottom plates containing 6 × 10⁴ cells in 100 µl of cell medium per well. Opsonized, ethidium bromide-stained merozoites (4–6 × 10⁵ per well) were washed twice with FACS buffer, resuspended in cell medium, and 100 µl were added to each leukocyte containing well. After an incubation of 30 min (unless stated otherwise) at 37 °C and 5 % CO₂, plates were centrifuged in a prechilled centrifuge and washed twice with ice-cold FACS buffer to stop phagocytosis. Cells were resuspended in 200 µl of cold FACS buffer and incubated for 1 hour at 4 °C with 1:1600 FITC antihuman CD14 (clone TuK4; Thermo Fisher Scientific MA1-82074), 1:400 BV786 antihuman CD16 (clone 3G8; BD Biosciences 563690), 1:800 APC antihuman CD45 (clone HI30; BD Biosciences 555485), and 1:800 BV421 antihuman CD66b (clone G10F5; BD Biosciences 562940) antibodies. After washing thrice with FACS buffer, sample fluorescence was quantified with a CytoFLEX S (Beckman Coulter Life Sciences). Phagocytosis was determined by measuring the ethidium bromide fluorescence using the 610/20 nm detector. Data analysis were performed with Kaluza Analysis Software version 2.1 (Beckman Coulter Life Sciences). The gating strategy used is shown in Supplementary Figure 2.

PBLs were distributed in 96-well U-bottom plates containing 5 × 10⁴ cells in 100 μl of cell medium per well. Then, 50 μl of cell medium with 12 μM of 2’,7’-dichlorodihydrofluorescin diacetate (DCFH₂-DA, 3 μM final concentration) and 1:200 dilution of surface staining antibodies (1:800 final dilution) were added. The staining antibodies were APC anti-human CD45 (clone HI30; BD Biosciences 555485), BV421 anti-human CD66b (clone G10F5; BD Biosciences 562940), and APC-AF750 anti-human CD14 (clone TuK4; Thermo Fisher Scientific MHCD1427) selective for leukocytes, granulocytes, and monocytes, respectively. Immediately, 50 μl of merozoites resuspended in cell medium and opsonized with immune or non-immune plasma diluted 1:100 was added to each well. After an incubation of 30 min at 37 °C, cells were washed thrice with FACS buffer (PBS with 0.5% BSA + 2 mM EDTA). Samples were quantified with a CytoFLEX S (Beckman Coulter Life Sciences) flow cytometer. DCF signal was determined by measuring the fluorescence using the 525/40 nm detector. Data analysis was performed with Kaluza Analysis Software version 2.1 (Beckman Coulter Life Sciences). The gating strategy used is shown in Supplementary Fig. 1.

The process for quantification of merozoite-phagocytosis has been described in detail^{10 (link)}. Leukocytes were distributed in 96-well U-bottom plates containing 5 × 10⁴ cells in 100 μl of cell medium per well. Ethidium bromide-stained merozoites were resuspended in cell medium, opsonized with immune and non-immune plasma at a 1:100 dilution, and 100 μl were added to each well. After a 30 min incubation at 37 °C, cells were washed thrice with cold FACS buffer. Cells were resuspended in cold FACS buffer containing the following antibodies diluted 1:800: BV421 anti-human CD66b (clone G10F5, BD Biosciences 562940); APC anti-human CD45 (clone HI30, BD Biosciences 555485); and APC-AF750 anti-human CD14 (clone TuK4, Thermo Fisher Scientific MHCD1427) (Supplementary Fig. 1). After a 30 min incubation at 4 °C, cells were washed thrice with cold FACS buffer. Samples were quantified with a CytoFLEX S flow cytometer and analyzed with Kaluza Analysis Software version 2.1. Phagocytosis was measured using the 610/20 nm detector to quantify the ethidium bromide signal.