Splenocytes were obtained from the spleens of DBA/1 mice at autopsy (on day 52 or 53 after CII immunization). An Fc-blocking antibody was used to prevent nonspecific binding (anti-mouse CD16/32; BioLegend, San Diego, CA, USA). Splenocytes were stained with peridinin chlorophyll protein complex-conjugated anti-mouse CD45 (1.25 μL/well, BioLegend), allophycocyanin-conjugated anti-mouse CD3e (1 μL/well, eBioscience, San Diego, CA, USA), fluorescein isothiocyanate conjugated anti-mouse CD4 (2 μL/well, BD Biosciences), and phycoerythrin (PE)-cyanine7-conjugated anti-mouse CD8a (0.5 μL/well, eBioscience). The fraction of CD138-positive cells was analyzed (PE rat anti-mouse CD138, 1 μL/well, BD Biosciences) as described previously [24 (link)].
The macrophage subset, the proportion of proinflammatory M1 macrophages (CD45+CD64+CD11c+CD206−) and anti-inflammatory M2 macrophages (CD45+CD64+CD11c−CD206+), was also analyzed as described previously [25 (link)].
The macrophage subset, the proportion of proinflammatory M1 macrophages (CD45+CD64+CD11c+CD206−) and anti-inflammatory M2 macrophages (CD45+CD64+CD11c−CD206+), was also analyzed as described previously [25 (link)].
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