Pierce fab micro preparation kit

For Fab‐PLA, the PLA-probes were prepared as previously described (44 (link)). F(ab)-fragments were prepared from the corresponding antibodies using the Pierce Fab Micro Preparation Kit (Thermo Fisher). After buffer exchange (Zeba spin desalting columns; Thermo Fisher), F(ab)-fragments were coupled with PLA probemaker (Sigma‐Aldrich). The cells were activated with 1 mM freshly prepared pervanadate or treated with RTX and then fixed for 20 min with 4% paraformaldehyde. Details about PLA-probes are provided in SI Appendix, Methods and Dataset S4.

Coating antibodies of the initial MPO-DNA complex ELISA protocol were processed with Pierce™ Fab Micro Preparation Kit (Thermo Fisher Scientific) to Fab and Fc fragments according to the manufacturer’s recommendations. First, 125 μg of antibody in a volume of 125 μl were digested with 65 μl of agarose-immobilized papain in a spin-column (kit components) at 37°C for 5 h under constant mixing. The sample was separated from the immobilized papain by double centrifugation at 5000 x g for 1 min. To further fraction the generated antibody fragments the solution was added to an equilibrated protein A column (kit component) and after 10 min of incubation, Fab fragments were retrieved by a centrifugation step at 1000 x g for 1 min (Fab fragment fraction 1). Furthermore, Fab fragment fractions 2 and 3 were obtained by washing the column twice with 200 μl of PBS and centrifuging again. Fc fragments bound to protein A (Fc fragment fractions 1, 2 and 3) were comparably collected by adding 400 μl of elution buffer to the column and centrifuging. 40 μl of neutralization buffer (kit component) were immediately added to each Fc fragment fraction. Protein content after purification was quantified using a Nano Drop 8000 spectrophotometer (Thermo Fisher Scientific).

Splenocytes from three age-matched male mice immunized with sheep red blood cells for 11 days were pooled. B cells were enriched by magnetic CD43-depletion (Miltenyi Biotech) and stained with rat anti-mouse IgG1-FITC Fab fragments (clone A85-1, BD catalogue no. 553443). Fab fragments were generated by papain cleavage using the Pierce Fab Micro Preparation Kit (Thermo Scientific) according to the manufacturer's instructions. Subsequently, cells were loaded with Indo-1-AM as previously described16 (link). mIgG–BCRs were stimulated with 20 μg ml⁻¹ goat anti-mouse IgG F(ab')₂ fragments (Jackson Immuno Research). For analysis of mIgG1-expressing cells, FITC-positive cells were gated.

For Fab‐PLA, F(ab′)₂ fragments were prepared from the corresponding antibodies using the Pierce Fab Micro preparation Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. In brief, F(ab′)₂ fragments were desalted (Zeba™ spin desalting columns, Thermo Fisher Scientific) and coupled with PLA Probemaker Plus or Minus oligonucleotides according to the manufacturer's protocol (Sigma‐Aldrich) to generate Fab‐PLA probes. For in situ PLA experiments, the cells were allowed to attach to polytetrafluoroethylene (PTFE)‐coated slides (Thermo Fisher Scientific) for 30 min at 37°C. Depending on the experiment, the cells were activated or treated with inhibitors and then fixed for 20 min with 4% paraformaldehyde in PBS. PLA was performed as previously described (Klasener et al, 2014). In brief, after incubation with a blocking solution containing 25 μg/ml sonicated salmon sperm DNA and 250 μg/ml bovine serum albumin (BSA) in PBS, the cells were incubated with Fab‐PLA probes in PBS. PLA signal amplification was performed following the manufacturer's protocol. The resulting samples were directly mounted on slides with DAPI‐Fluoromount‐G (Southern Biotech) to visualize the PLA signals in relation to the nucleus.

UCHT1 Fab fragments were prepared using the Pierce^® Fab Micro Preparation Kit from Thermo Fisher Scientific, which uses the enzyme papain to cleave the complete UCHT1 antibody and protein A coupled beads to purify the Fab fragments. The purified Fab fragments were analyzed by SDS-PAGE and Coomassie staining. To generate reduced Fab fragments (Fab_red) the purified Fab fragment was incubated with 10 mM dithiothreitol for 30 min at room temperature. Afterward, 1 mM iodoacetamide was added and incubated for further 30 min at room temperature. The Fab_red were then immediately used. Purity of Fab and Fab_red was further tested by their inability to induce Ca²⁺ influx in T cells.

Human anti-gp120 monoclonal antibody 2G12 was purchased from Polymun Scientific, Austria. 2G12 Fab fragments were generated using the Pierce Fab Micro Preparation kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions. Anti-human IgG Fab fragments (Jackson ImmunoResearch Europe, Ely, UK) were coupled to Abberior STAR RED (KK114) dye (Abberior GmbH, Göttingen, Germany) via NHS-ester chemistry according to the dye manufacturer’s instructions. In addition, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids, Alabaster, AL, USA. Abberior STAR RED (KK114)–1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (KK114-DPPE) was purchased from Abberior GmbH, Göttingen, Germany [10 (link)].