Moflo cell sorter

Single-cell suspensions were prepared from the spleens and lymph nodes of donor CD45.1⁺ or CD45.2⁺ mice and CD4⁺ T cells were enriched using immunomagnetic positive selection (StemCell Technologies), at >96% purity. A total of 1 × 10⁶ CD4⁺ T cells were injected into CD45.1⁺CD45.2⁺ recipients via the tail vein. Where indicated, enriched EF4.1 CD4⁺ T cells were further purified (>98% purity) by cell sorting, performed on MoFlo cell sorters (Dako-Cytomation, Fort Collins, CO, USA).

The constructed plasmids, linearized with SnaBI, were transfected into 1 × 10⁷ EacWT or EteWT sporozoites by restriction enzyme-mediated integration as previously described^{39 (link)}. After nucleofection (Program U-033, AMAXA, Switzerland), the transfected E. tenella sporozoites were inoculated into the ileocecal opening via the cloaca, and the transfected E. acervulina sporozoites were inoculated intravenously into the wing vein of five 1-week-old chickens equally for stable transfection^{23 (link)}. The transgenic oocysts were selected in chickens by the MoFlo^® Cell Sorter (Dako-Cytomation, Fort Collins, CO) on the single-cell mode in vitro, and/or 150 mg/L pyrimethamine (Sigma-Aldrich Co., St. Louis, Mo., USA) press by drinking water in vivo, then inoculated into coccidian-free chickens for the propagation of next generation oocysts.

1 × 10⁸ LCL yielded approximately 6 × 10⁶ G₂M cells from a 4-h sort. Cells were treated with 0.1 µg/ml colcemid for 4 h to enrich the mitotic fraction. Cells were then harvested by centrifugation and washed three times in PBS. Ice cold acetone was added dropwise to ~ 1 × 10⁷ cells/ml on a very slow vortex before freezing to − 20 °C for at least 2 h. Cells were then centrifuged at 30×g for 10mins, 4 °C (MSE chillspin), acetone was carefully removed from the pellet and ice-cold PBS was added dropwise to ~ 1 × 10⁷ cells/ml. The cells were centrifuged and resuspended in PBS at 2 × 10⁷ cells/ml. Propidium iodide was added to a concentration of 10 µg/ml and incubated for 30 min at 4 °C. Cells were sorted into G₁ and G₂M fraction based on Propidium iodide fluorescence using a MoFlo cell sorter (DakoCytomation).

Volunteers from a Phase Ia safety and immunogenicity clinical trial were bled seven days after the second immunization using MVA (modified vaccinia virus Ankara) encoding PfRH5FL (Payne et al., 2017 ). Blood was collected from volunteers in heparinized tubes and centrifuged in Leucosep tubes (Greiner Bio one) to separate the peripheral blood mononuclear cells (PBMC). The PBMC were enriched for B cells using a human pan-B cell enrichment kit (StemCell Technologies, Inc.) and resuspended in DMEM before staining with a CD19⁺, CD10^–, CD21^–, CD27⁺, CD20^–, CD38⁺, IgG⁺ fluorophore-conjugated antibody panel. Plasmablasts were single-cell sorted using a MoFlo cell sorter (Dako cytomation) into 96-well PCR plates containing 10 μL of 10 mM Tris HCl buffer containing 40 U/mL of RNase inhibitor (Promega). The study received ethical approval from the Oxfordshire Research Ethics Committee A in the UK (REC reference 14/SC/0120). The volunteers signed written consent forms and consent was verified before each vaccination.

Synovial fluid was incubated with 1000 U ml⁻¹ hyaluronidase (Hyalase^TM, Wockhardt UK Ltd) at 37 °C for 15 min to reduce viscosity. Mononuclear cells were isolated from SF using density gradient centrifugation and from mechanically dissociated ST as previously described [28] (link). Cells from 4 SF and 2 ST samples (Suppl table 1; patients 1–6) [27] were stained for flow cytometry with mouse monoclonal antibodies against CD19 (Biolegend 302234) and FcRL4 (Biolegend 340204). A total of 96 single B cells from CD19+FcRL4+ and CD19+FcRL4- populations were sorted from each SF and ST samples using a MoFlo cell sorter (Dako) into 96-well PCR plates as previously described [29] (link), [30] (link). Purity of sorted cells was in excess of 95%.
For detection of IgA B cell receptors, mononuclear cells were isolated from SF of 11 further RA patients (Suppl. table 1; patients 7–17) [27] . Surface receptor-bound antibodies were first removed by incubating SF B cells in 0.1 M glycine buffer at pH3 for 90 s. Cells were washed, labeled with antibodies to IgA (Miltenyi), FcRL4 (Biolegend) and CD19 (Biolegend) and analyzed on a Cyan ADP flow cytometer (Beckman).