Pe labeled anti human igd

IgM or IgG memory B cells were isolated from frozen peripheral blood mononuclear cells (PBMCs) by magnetic cell sorting with 0.5 μg ml⁻¹ anti-CD19-PECy7 antibodies (BD, 341113) and mouse anti-PE microbeads (Miltenyi Biotec, 130-048-081), followed by FACS sorting using 3.75 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch, 109-606-170), 5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgM (Invitrogen, A21215) and 1/40 PE-labeled anti-human IgD (BD, 555779). As previously described^{47 (link)}, sorted B cells were immortalized with Epstein-Barr virus (EBV) and plated in single cell cultures in the presence of CpG-DNA (2.5 μg ml⁻¹) and irradiated PBMC-feeder cells. Two weeks post-immortalization, the culture supernatants were tested (at a 2/5 dilution) for binding to PfSPZ by flow cytometry using a no-wash protocol. Briefly, cryopreserved PfSPZ were thawed, stained with the supernatants in 6.25× SYBR Green I for 30 min at room temperature, and incubated with 2.5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG or anti-human IgM for 1 hour at 4°C. Only supernatants that did not bind to control beads were selected to exclude polyreactive antibodies.

IgM or IgG memory B cells were isolated from frozen peripheral blood mononuclear
cells (PBMCs) by magnetic cell sorting with anti-CD19-PECy7 antibodies (BD, 341113) and
mouse anti-PE microbeads (Miltenyi Biotec, 130-048-081), followed by FACS sorting using
goat Alexa Fluor 647-conjugated anti-human IgG (Jackson ImmunoResearch, 109-606-170) or
anti-human IgM (Jackson ImmunoResearch, 109-606-129) and PE-labeled anti-human IgD (BD,
555779). As previously described18 (link), sorted B cells
were immortalized with Epstein-Barr virus (EBV) and plated in single cell cultures in the
presence of CpG-DNA (2.5 µg ml⁻¹) and irradiated PBMC-feeder
cells. Two weeks post-immortalization, the culture supernatants were tested (at a 2/3
dilution) for the presence of LAIR1-containing antibodies using the bead-based immunoassay
described above. For several donors, the culture supernatants were also tested for the
ability to bind to IEs from a mixture of four parasite isolates (3D7-MGD21⁺,
9106, 9605 and 11019) by flow cytometry. Briefly, cryopreserved IEs were thawed, stained
with 10× SYBR Green I for 30 min at room temperature, and incubated with the B-cell
supernatants for 1 hour at 4°C. Detection of antibody binding was done with 2.5
μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG.