Captureselect fcxl affinity matrix

To capture IgG-BCRs, 20 million Ramos B cells were washed with PBS, to remove FCS from the cell culture medium, and lysed in 8 ml PBS + 1% Triton-X100 for 60 min at 37 °C. IgG-BCRs were captured from cell lysis supernatants using 20 µl CaptureSelect^TM FcXL Affinity Matrix (Thermo Fisher Scientific) and an overnight incubation at 4 °C. B cell secreted IgG were captured from 12 ml Ramos B cell supernatant (cultured at a density of two million cells/ml) by a 4 °C overnight incubation with 20 µl CaptureSelect^TM FcXL Affinity Matrix (Thermo Fisher Scientific). Laemmli sample buffer (4×) (Bio Rad) was added to the IgG/FcXL bead slurry, boiled for 5 min at 95 °C, and loaded on a 4 to 15% SDS gel (Bio Rad). Proteins were detected with Coomassie Brilliant Blue G-250 Dye (Thermo Fisher Scientific) or the SilverQuest^TM Silver Staining Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The size was determined using the PageRuler^TM Plus Prestained Protein Ladder (Thermo Fisher Scientific).

For inhibitor or drug tests, most chemicals were reconstituted in DMSO and diluted with 0.2% BSA medium. However, MβCD was directly dissolved in the 0.2% BSA medium. These samples were then applied during the serum starvation period prior to the application of flow. The following concentrations and incubation times were utilized for each chemical: LDN193189 (1 µM; Cat: S2618, Selleck Chemicals, Houston, TX, USA) and Y27632 (10 µM; Cat: 688000, Calbiochem, San Diego, CA, USA) were applied for 60 min prior to flow. Latrunculin B (0.015 µM; Cat: 428020, Calbiochem), MβCD (5 mM; Cat: 377110050, Thermo Fisher Scientific), and Dynasore (100 µM; Cat: D7693, Sigma-Aldrich) were applied for 30 min prior to flow. Controls were treated with the same percentage of DMSO in 0.2% BSA medium as the inhibitor or drug-treated samples: LDN193189 control, 0.2% DMSO; Y27632 control, 0.1% DMSO; Latrunculin B and Dynasore controls, 0.4% DMSO; MβCD control, 0% DMSO. ALK1-Fc (Cat: 370-AL, R&D Systems) was incubated at a final concentration of 1.25 mg/mL with 30% BSA on an orbital shaker at 4 °C overnight. ALK1-Fc-complexes were then captured with CaptureSelect FcXL Affinity Matrix (Cat: 194328010, Thermo Fisher Scientific). The ALK1 ligand-depleted 30% BSA was diluted to 0.2% with phenol red-free EBM2 for use.

The cDNA fragment encoding amino acids 18–541 of hemagglutinin (HA) from influenza A H1N1 (A/Puerto Rico/8/1934 (PR8)) was used to generate IgG1 Fc fragments with HA fused to the N-terminal end of one of the HCs. The HA cDNA was subcloned into the pFUSE-hIgG1-Fc2 expression vector (InvivoGen) to generate pFUSE-HA(PR8)-hIgG1-Fc2. Removal of the multiple cloning sites in the target vector generated pFUSE-naked-hIgG1-Fc2. Mutagenesis was then performed to introduce the knob-in-hole mutations. The “knob” mutation (T366Y) was introduced into pFUSE-HA(PR8)-hIgG1-Fc2, while the “hole” mutation (Y407T) was introduced into the pFUSE-naked-hIgG1-Fc construct either alone or in combination with the REW substitutions. The monovalent Fc fusions were produced in Expi293 cells by transient transfection adding a 2:1 ratio of the knob:hole constructs per the manufacturer instructions. The fusions were purified using a CaptureSelect FcXL affinity matrix (ThermoFisher) packed in a 5 ml column (Repligen) per the manufacturer recommendations. Eluted fractions were collected, concentrated and buffer exchanged to 1xPBS using Amicon Ultra-30 spin columns (Merck). Monomeric fractions of the monovalent fusions were then isolated by SEC using a Superdex 200 Increase 10/300 GL column (Cytiva Life Sciences) with an ÄKTA Avant 25 instrument (Cytiva Life Sciences).