At selected time points following immunization, whole spleens were mechanically dissociated to generate single-cell suspensions. ACK lysis buffer was used to deplete red blood cells, after which splenocytes were resuspended in FACS buffer (2% FBS in PBS), incubated with Fc-block (clone 2.4G2, BD Biosciences) and stained for viability with Live/Dead Blue viability dye (Thermo Fisher Scientific) for 20 min at 4°C. Cells were then stained with a variety of antibodies, including antibodies against CD4-APC-eF780, CD8-APC-eF780, Gr-1-APC-eF780, F4/80-APC-eF780, B220-V421, CD95-PE-Cy7, CD38-A700, CD45.1-PerCP-Cy5.5, CD45.2-PE, IgD-APC-Cy7, and C138-BV650. For surface staining of eOD-GT8 binding, probes were labeled as described previously and added 30 minutes prior to antibody staining (Wang et al., 2021a (link)). Cells were acquired by a BD LSRFortessa (BD Biosciences) for flow cytometric analysis and sorted using a BD FACS Aria II instrument (BD Biosciences). Data was analyzed using FlowJo software (Tree Star).
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