Quadromacs

Human monocytes were isolated from 500 ml fresh whole blood (drawn within 1 h before use) of healthy male donors. Blood was layered onto an equal volume of 1-Step Polymorphs (Accurate Chemical & Scientific Corporation, USA) and centrifuged at 650 × g for 35 min. After centrifugation, the peripheral blood mononuclear cells (PBMCs) were collected, and normal osmolarity was restored by adding an equal volume of 0.45% cold NaCl. After erythrocyte lysis using a hypotonic buffer, cells were washed twice in cold PBS and counted using a Neubauer chamber. Cell viability of >95% was assessed by trypan blue staining. Monocytes were isolated from the PBMCs using the monocyte isolation kit II and quadro-MACS (Miltenyi Biotec, UK), following manufacturer’s instructions. Purity of the monocytes (>90%) was assessed as described in Klassert et al.32 (link).

LCMV Armstrong was kindly provided by S.J. Ha (Yonsei University, Seoul, Korea). Naive CD8⁺ subsets were purified from pulled spleens and LNs (or from thymus) of indicated mice and transferred to B6 hosts (10²–10⁴ cells/mice). In some experiments, two subsets were co-transferred using different congenic markers. Mice were infected with LCMV (2 × 10⁵ pfu/mice) day after transfer and assessed 6–8 days later unless indicated otherwise. Spleens and LNs were harvested and analyzed by flow cytometry. To analyze antigen-specific CD8⁺ T cells from B6 donors, two-step enrichment method was used. Thy1.1 B6 mice were specifically used as hosts for this experiment. Spleens and LNs were pulled and stained with biotin-conjugated anti-CD4 and anti-B220 antibodies (Invitrogen), followed by anti-biotin MicroBeads (Miltenyi Biotec). Stained cells were purified using LS Column and QuadroMACS (Miltenyi Biotec) as described elsewhere. Flow-through was harvested and stained with biotin-conjugated anti-Thy1.1 antibody (Invitrogen), followed by anti-biotin MicroBeads. Cells were purified and the flow-through, now enriched with donor cells, were harvested and stained with GP33- or NP396-tetramers.

Frozen viable pleural effusion or ascites vials were thawed and CD45+ white blood cells were depleted using Miltenyi’s QuadroMACS. Individual cells were captured using Fluidigm C1 chips (10–17 μm cell diameter); whole-genome amplification was performed per manufacturer’s instructions. Targeted amplification of mutation-containing regions was performed using Fluidigm Access Array and BioMark instruments per manufacturer’s instructions. Libraries were sequenced on Illumina MiSeq. Reads were aligned to hg19 with BWA MEM v0.7.8. Variants were called using MuTect v1.1.4. Co-occurrence of mutations (at least 1 mutant read) in individual cells was evaluated using Fisher’s exact test. Single cells with more than one subclone-defining mutation are shown.

Splenocytes were isolated as described earlier (Farooque et al., 2014a (link)) and were sorted using biotin-labeled anti-mouse GR-1-coated protein A beads and the QuadroMACS™ (Miltenyi Biotec) separator (Horgan et al., 2001 ). Briefly, anti-GR-1 antibody (BioLegend) labeled magnetic Protein beads (Miltenyi Biotec) were incubated with splenocytes according to the manufacturer’s protocol and washed with MACS buffer (Horgan et al., 2001 ). Isolated GR-1⁺ PMNCs were stained with CFSE (Thermo Scientific) as described in Milanez-Almeida et al. (2015 (link)) and administered (1 × 10⁶ cells/100 μL/mice) to animals in the various groups via the tail veins. Cell localization was observed using the FPRO Imaging Station (Carestream Molecular Imaging, United States).

The P. falciparum 3D7 wild type cell line and the 3D7_SLI_slp-gfp_glmS (Slp) strain were cultured in 0+ human red blood cells using RPMI 1640 medium (Sigma) with added 0.2 mM hypoxanthine, 25 mM HEPES, 0.5% Albumax and 12.5 μg/ml Gentamicin (cRPMI) at a hematocrit of 2.5% and an average parasitemia of 1–5%. For washing of cells, cRPMI without Albumax was used (iRPMI). For Slp, 600ug/ml Geneticin-G418 (Thermo Fisher) was added. Parasites were incubated at 90% relative humidity, 5% O₂ and 3% CO₂ at 37°C. Parasites were synchronized by selective lysis of schizonts using 5% D-sorbitol for 10 minutes at 37°C, performed three times throughout one week or alternatively via quadroMACS (Miltenyi Biotec) magnetic purification of late stage schizonts followed by sorbitol treatment.

For analysis of parasite DNA content via flow cytometry cells were first synchronized using quadroMACS (Miltenyi Biotec) magnetic purification of late stage schizonts followed by sorbitol treatment after 3h. iRBCs with a parasitemia of ~0.5% were thereafter treated with 3.5mM GlcN or left untreated and incubated at the beforementioned incubation conditions, after 24 hpi 200μl of medium was taken and fixed with 4%PFA/PBS and 0.0075% Glutaraldehyde for 12h at 4°C every 3h until 54 hpi. Samples were washed, permeabilized with Triton X–100 (Sigma, T8787) for 8 min and treated with 0.3mg/ml Ribonuclease A (Sigma, R4642) in PBS for 30 min. Cells were washed twice and resuspended in 100μl staining solution (1:2000 SYBR green I in PBS) followed by 20 min incubation. After subsequent washing cells were diluted to the preferred dilution and DNA content was analyzed via flow cytometry. Cells were gated for 2000 single, infected red blood cells and SYBR green emission intensity after FITC excitation was measured for each time point and condition. Intensity was normalized to equal starting emission in each individual replicate. For determination of DNA content >2, we quantified the amount of iRBCs >2N relative to the total number of iRBCs. Graphs were plotted using Prism GraphPad. N = 3.

PBMC were isolated by density gradient centrifugation (histopaque; Sigma life science). CD14⁺ monocytes were purified by positive selection using anti-CD14 monoclonal antibody-coated magnetic microbeads according to the manufacturer’s instructions (Quadro Macs; Miltenyi Biotech). CD14⁺ monocytes were cultured in T-225 flasks (Corning Incorporated, Costar) at 6 x 10⁵ cells per ml in Advanced RPMI 1640 medium (Gibco, Life Technologies) supplemented with 10% heat-inactivated bovine serum (Quality Biologicals), 2 mM l-glutamine (Invitrogen), 0.05 mM β-mercaptoethanol, 50 ng/ml granulocyte-macrophage colony-stimulating factor (R&D Systems), 16 ng/ml interleukin-4 (R&D Systems), 200 IU/ml penicillin, and 200 μg/ml streptomycin sulfate (Invitrogen). The cells were incubated for 7 days at 37°C, 5% CO₂ as described previously [50 (link)].