Apc efluor 780 clone 30 f11

For both malaria infections and IVET assay, 2 μl of whole blood samples were stained with 2 μg/ml streptavidin-PE-Cy7 (only for experiments with biotinylated erythrocytes), 1 μg/ml anti-CD45–allophycocyanin (APC)–eFluor 780 (clone 30-F11), 1 μg/ml anti-CD71 (TFR1)–PerCP–eFluor 710 (clone R17217) (eBioscience, San Diego, CA), 4 μM Hoechst 33342 (Sigma-Aldrich, St Louis, MO) and 12 μM JC-1 (Thermo Scientific, Waltham, MA) in MTRC. All samples analysed through flow cytometry were performed on BD LSRFortessa (BD Biosciences, Franklin Lakes, NJ), where 100,000 to 2,000,000 events were collected and visualized on FACSDiva^TM and FlowJo software. The RBCs and leukocytes were first selected on forward scatter and side scatter channels (FSC/SSC) signals, followed by gating of single cells based on FS area to height ratio. RBCs were further isolated by gating on CD71 negative and CD45 negative population, followed by gating on Atto-labelled and Biotin-labelled erythrocytes on appropriate channels (APC for Atto-633, PE for Atto-565 and PE-Cy7 for Biotin). The parasitemia of each labelled erythrocyte population was determined by gating on Hoechst 33342 positive and JC-1 positive population.

The mesenteric lymphoid nodes were carefully mashed with wet slides to decrease friction and the solutions were filtered through a 70 μM cell strainer. Cells were isolated, counted and plated on anti-CD3 (clone17A2, eBioscience, San Diego, CA, USA) and anti-CD28 (clone 37.51, eBioscience)—coated plates for FcγR blocking. Then, the cells were transferred to polystyrene tubes for surface staining with anti-CD4 (PerCP-Cy™5.5, clone RM4-5 BD Pharmigen™, Franklin Lake, NU, USA), anti-CD45 (APC-eFluor®780, clone 30-F11, eBioscience) and Viability Dye (eFluor®660, eBioscience) for 15 min at 4°C in the dark. The cells were then fixed, permeabilized with the Fixation/Permeabilization kit (eBioscience) and intracellular staining was done with anti-Foxp3 (PE, clone FJK-16s, eBioscience) for 30 min at 4°C in the dark. Data collection was performed using a flow cytometer CANTO II (BD Biosciences San Diego, CA, USA).