Pe cyanine7 conjugated anti mouse cd8a

Single cell suspensions were obtained from the spleens of NZB/W F1 mice at autopsy (at 43–44 weeks of age). The splenocytes were stained with PerCP-Cy5.5-conjugated anti-mouse CD3e (PerCP-cy5.5-CD3e, eBioscience, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (FITC-CD4, BD Biosciences, San Jose, CA, USA), PE-cyanine7-conjugated anti-mouse CD8a (eBioscience), allo-phycocyanin (APC)-conjugated anti-mouse CD25 (BD Biosciences) and PE-conjugated anti-mouse CD138 (BD Biosciences).
T cell profile was analyzed as described previously^{2 (link)}; briefly, we examined proportions of Th1 cells (CD4⁺CD25⁺T-bet⁺), Th2 cells (CD4⁺CD25⁺GATA-3⁺), Th17 cells (CD4⁺CD25⁺ROR-γt⁺), and Treg cells (CD4⁺CD25⁺Foxp3⁺). To analyze T helper subsets, the splenocytes were stained with antibodies to CD4 and CD25 (FITC-conjugated anti-mouse CD4 and APC-conjugated anti-mouse CD25, BD Biosciences). Cells were fixed and permeabilized prior to staining with T-bet, GATA-3, ROR-γt and Foxp3 antibodies (PE-, BD Biosciences).

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)].