Single-cell suspensions were prepared by homogenising spleens through 100 m strainers and lysing erythrocytes using RBC lysis buffer (eBioscience). Fc receptors were blocked using anti- CD16/32 antibody (BD Pharmigen or in-house). T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCR-APC-Cy7 (H57-597), CD45.1-FITC (A20), V2-FITC (B20.1), V12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1- APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)- PE (CXCR3-173), IFN-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8) and Tbet-eFluor660 (4-B10) (eBioscience). Dendritic cells and monocytes were stained with the following antibodies (Biolegend): CD11c-Percp-Cy5.5 (N418), MHCII (1-A/1/E)-APC (M5/114-15.3), B220-AlexaFluor700 (RA3-6B2), TCR-APC-Cy7 (H57-597), Ly6c-FITC (HKJ.4), CD11b-BV421 (M1/70), Ly6g-PE (IA8), CD8-PE-Cy7 (53-6.7) and CXCL9-PE (MIG-2F5.5). Intracellular staining for IFN, Tbet, Bcl6, Ki67 or CXCL9 was performed with the eBioscience FoxP3 intracellular kit. Intracellular staining for p-S6 was performing using a monoclonal antibody (D57.2.2E), or respectively isotype control (Cell      22 Signaling Technology) with Cell Signaling Buffer Set A (Miltenyi Biotech) according to manufacturer's protocol. Cells were sorted using a MoFlo XDP (Beckman Coulter) or a FACSAria II (Becton Dickinson) instrument. Activated PbTII T cells were sorted as CD4+TCR+ and CD69+ and/or divided at least once as measured using the CellTrace Violet proliferation dye. Dendritic cells were sorted as CD11chiMHCIIhiTCR-B220-. Naive dendritic cells were further sorted as CD8+CD11b- or CD8-CD11b+. Inflammatory monocytes were identified as CD11bhiLy6ChiLy6GloTCR-B220-. Plasmodium chabaudi chabaudi AS parasites were used after one in vivo passage in WT C67Bl/6 mice. Mice were infected with 105 pRBCs i.v. and blood parasitemia was monitored by Giemsa-stained thin blood smears obtained from tail bleeds. Single cell capture and processing was performed using the Fluidigm C1 system as in (40). The cell suspension obtained from sorting was loaded on to the Fluidigm C1 platform using small sized capture chips (5-10m cells). 1 l of a 1:4000 dilution of External RNA Control Consortium (ERCC) spike-ins (Ambion, Life Technologies) was included in the lysis buffer. Reverse transcription and preamplification of cDNA were performed using the SMARTer Ultra Low RNA kit (Clontech). The sequencing libraries were prepared using Nextera XT DNA Sample Preparation Kit (Illumina), according to the protocol supplied by Fluidigm (PN 100-5950 B1). Libraries from up to 96 single cells were pooled and purified using AMPure XP beads (Beckman Coulter).
Experimental Factor: 2
time
Experimental Factor: Plasmodium chabaudi chabaudi AS
infection