August 29, 2021
and Z.L.B. respectively. Draining LNs from DEL-OVA immunized and peripheral LNs from unimmunized recipients were analyzed 2 weeks after transfer. GL7? Hy10 (left, middle panels) and endogenous (right panels) B cells were gated as in A. Representative of n = 2 independent experiments with 3C4 mice. D, Example of memory subpopulation gating from 18 week timepoint. Representative of n = 4 independent experiments with 6C12 mice. E, Plasma cell gating strategy. PCs were identified as B220lo intracellular Ighi cells (upper panels, red gates) that were larger and stained more brightly for intracellular Ig than B220+ cells (middle panels, black gates). Ig+B220+ cells (grey gates) shown for comparison. Class switched PCs were defined based on intracellular IgM staining (lower panels).(PDF) pone.0183877.s001.pdf (557K) GUID:?BE3F2C3E-4860-4A77-A5BB-AF85216DF34C S2 Fig: Single acquisition of threshold activating amount of Ag enables generation and persistence of memory B cells for 5 minutes at 37C, washed four times with room temperature DMEM supplemented with 4.5 g/L glucose, L-glutamine and sodium pyruvate, 2% FBS, 10 mM HEPES, 50 IU/mL of penicillin, and 50 g/mL of streptomycin, and transferred i.v. to recipient mice. Flow cytometery Single-cell suspensions from spleens or draining inguinal lymph nodes (dLNs) were incubated with biotinylated antibodies (S1 Table) for 20 minutes on ice, washed twice with 200 l PBS supplemented with 2% FBS, 1 mM EDTA, and 0.1% NaN3 (FACS buffer), incubated with fluorophore-conjugated antibodies and streptavidin (S1 Table) for 20 minutes on ice, washed twice more with 200 l FACS buffer, and resuspended in FACS buffer for acquisition. For intracellular staining, surface-stained cells were fixed Buclizine HCl and permeabilized for 20 minutes on ice with BD Cytofix/Cytoperm buffer, washed twice with 200 l BD Buclizine HCl Perm/Wash buffer, incubated with for 20 minutes on ice with fluorophore-conjugated antibodies (S1 Table), followed by two washes with 200 l Perm/Wash buffer, and resuspended in FACS buffer for acquisition. Data were acquired on a FACSCanto or LSRFortessa and analyzed using FlowJo (TreeStar). Statistics Statistical tests were performed as indicated using Prism 6 (GraphPad). Differences between groups not annotated by an asterisk did not reach statistical significance. No blinding or randomization was performed for animal experiments, and no animals or samples were excluded from analysis. Results To determine the ability of B cells to Buclizine HCl differentiate into various subpopulations of memory B cells after a single transient acquisition of Ag, and to define how their development and persistence over time depends on the dose of initially acquired Ag and reacquisition of Ag for 5 minutes with either a saturating (50 g/mL) or threshold activating (0.5 g/mL) concentration of the moderate affinity Ag duck egg lysozyme (DEL)  fused to ovalbumin (DEL-OVA) and the Buclizine HCl unbound Ag was then washed off. 105 Ag-pulsed Hy10 B cells were transferred into recipient mice, which had been s.c. immunized with OVA in CFA three days earlier to activate endogenous OVA-specific helper T cells. Under these conditions, DEL-OVA-primed B cells could not reacquire cognate Ag for 5 min with 0.5 or 50 g/mL DEL-OVA, were transferred into recipient mice s.c. preimmunized with OVA, DEL-OVA, or BSA in CFA. B, C, Expansion of Hy10 cells in recipient mice 2 weeks after transfer. Total (B) and GL7? (C) unswitched (left), and isotype-switched (right) Hy10 cells from LNs of recipient mice, shown as fraction of B220 normalized to the number of Hy10 cells transferred. n = 2 independent experiments with 3C6 mice. DCG, Memory B cell responses of unpulsed (open symbols) and 50 g/mL (filled symbols) or 0.5 g/mL (shaded symbols) DEL-OVA pulsed Hy10 B cells in draining inguinal LNs (dLNs, D, E) and spleens (F, G) of OVA (blue symbols), DEL-OVA (red symbols), and BSA (black symbols) immunized recipient mice 2 weeks (left panels), 4 weeks (middle panels) hJAL and 18 weeks (right panels) after transfer. DN, SP, and DP subpopulations gated as in S1A and S1C Fig and shown as ratio to total B220+CD4?CD8? singlet lymphocytes. H, Hy10 PC recall response in dLNs 3 days after secondary Buclizine HCl s.c. immunization with 50 g DEL-OVA in IFA, 18 weeks after initial transfer of Hy10 cells. For 2 and 4 week timepoints and recall, n = 2 independent experiments with 4C6 mice per condition; for 18.