Pe anti human igg fc antibody

Crystals (100 µg) were incubated in 50 µl serum or HBSS + 10% BSA for 30 min at 37 °C with mild agitation. Crystals were harvested by centrifugation at 1000×g for 2 min and washed with HBSS. IgM and IgG were detected using PE anti-human IgM antibody (#314508, BioLegend) and PE anti-human IgG Fc antibody (#410708, BioLegend) (both diluted 1:25 in HBSS + 5% BSA), respectively. Particle fluorescence was analyzed using a FACS Canto II (BD Biosciences) and BD FACSDiva software version 6.1.3 (www.bdbiosciences.com). Flowing Software version 2.5.1 (Perttu Terho, University of Turku, Finland, https://bioscience.fi/services/cell-imaging/flowing-software/) was used for analysis of data obtained.
Fluorescence microscopy was performed as described before^{26 (link)} and images were acquired using an Olympus IX81 inverted microscope and CellR software (version 3.2; www.olympus-lifescience.com/en/software/). Brightness was adjusted, pseudo-color was inserted in the grayscale image, and scale bar was added using ImageJ (version 1.53e; https://imagej.nih.gov/ij/).

Flow-cytometry analysis was performed on a BD Biosciences LSR-II cytometer equipped with UV, violet, blue, and red lasers. Doublets and dead cells were excluded by forward scatter, side scatter, and use of the AF350 LIVE/DEAD fixable violet dead cell stain (Thermo Fisher, #L34964, 1:1000). B cells were tested for CD19 (BioLegend, #302243 Brilliant Violet 605 anti-human CD19 antibody, 1:200), CD3 (BioLegend, #317329, Brilliant Violet 785 anti-human CD3 antibody, 1:200), and CD38 (BD, #551400, PerCP-Cy5.5 mouse anti-human CD38, 1:200). SupT1 cells were tested for CD8 (BD, #560662, PerCP-Cy5.5 mouse anti-human CD8, 1:100). Cells were fixed with the Cytofix/Cytopermreagent and washed in Perm/Wash buffer (BD). Cells were then permeabilized using Permeabilization Plus Buffer (BD, #561651). B cells were subsequently stained intracellularly for IgG (BioLegend, #410707, PE anti-human IgG Fc antibody, 1:200) and IgM (BioLegend, #314532PE/Cyanine7 anti-human IgM antibody, 1:200), and SupT1 cells were tested for p24 (Beckman Coulter, #6604667, HIV-1 core antigen-RD1, KC57, 1:100).

PD-L1 positive aAPC/CHO-K1 (Cat # J1205) cells and PD-L1 negative aAPC/CHO-K1 (Cat # J1191) were purchased from Promega. Bjab cells were purchased from (ABC-TC514S, AcceGen, New Jersey, USA). Raji cells (Cat# CCL-86) and Jurkat cells (TIB-152) were purchased from American Type Culture Collection (ATCC). Both T cells and B cells were maintained in ATCC-formulated RPMI-1640 Medium (ATCC 30-2001) with 10% fetal bovine serum (ATCC 30-2020). To confirm the cell surface binding of isolated antibody binders, cells were treated with VH 1-16, VH 1-16-3, E1-2, G10 or m971 for 1 h at 4°C and then stained with anti-DYKDDDDK Antibody, FITC (130-127-934, Miltenyi Biotec), or anti-DYKDDDDK (flag tag) Antibody, PE(130-101-577, Miltenyi Biotec), or PE anti-human IgG Fc Antibody (410707, BioLegend) for 0.5 h at 4°C. After each incubation with antibodies, cells were washed with cold PBS. Data were acquired using the flow cytometry BD LSR II (San Jose, CA) and analyzed by FlowJo 10.7.1.