Anti mouse cd3e fitc

Whole kidneys and tissue from 1/3 of a spleen were processed to single cell suspensions and stained with the following antibodies purchased from BD Bioscience (San Diego, CA, USA): anti-mouse CD3e-FITC, Ly6c-FITC, CD11b-PE, CD11c-PE, CD45-PE, k Light Chain-PE, CD8a-PerCP, CD4-APC, CD11b-APC, CD138-APC, CD86-FITC. Also used for staining were anti-mouse MHC-II-FITC and CD103-APC (both from eBioscience, San Diego, CA) and F4/80-APC from Bio-Rad (Raleigh, NC, USA). Detached GEnCs were stained with annexin/PI (Miltenyi Biotec, Germany). The FACSCalibur flow cytometer was used for acquiring the cells and analysis was done using the CellQuest software.

Spleen cells were prepared as described previously [18 (link)]. Briefly, the spleen was mechanically dissociated and passed through a 70um filter. The final 4 ml suspension was layered onto 2 ml Ficoll-Paque (GE,17–1440-02) and the cells at the interface were collected after certification (500 g,20 min,4 °C). Single cell samples were incubated with antibodies to surface antigens for 30 minutes on ice at 4 °C in the dark. Fluorochrome compensation was performed with single-stained UltraComp eBeads. Flow cytometery was performed on the BD LSRFortessa flow cytometer (BD biosciences). Data analysis were performed using Flowjo software.
The antibodies used for profiling splenic T cells included anti-mouse CD45-Pacific Blue (1:200; BioLegend, 103,126); anti-mouse CD3e-FITC (1:200; BD Pharmingen, 553,062); anti-mouse CD4-APC-Cy7 (1:200; BD Pharmingen, 552,051); anti-mouse CD8-Percp (1:200; BioLegend, 100,732); anti-mouse CD44-V500 (1:200; BD Pharmingen, 560,781); anti-mouse CD62L-BUV395 (1:200; BD Pharmingen, 740,218); anti-mouse CCR2-PE (150609).

Spleen was harvested from sham mice to prepare single cell suspensions as we described above. CD8⁺ T cells were isolated using mouse CD8a microbeads (Miltenyi Biotec, 130–117-044) according to the manufacturer’s instructions. Coculture System was established as described before [18 (link)], isolated CD8⁺ T cells in a transwell insert were incubated with brain slices in the lower chamber in culture media (RPMI 1640, 10% FBS, 1% penicillin/Strepromycin, 1 mM pyruvate sodium, 55 μm β-mercaptoethanol with the presence of soluble anti-CD3, anti-CD28 and IL-2) for 24 h. Then cells in the lower compartment were collected and stained with anti-mouse CD3e-FITC (1:200; BD Pharmingen, 553,062), anti-mouse CD8a-APC (1:200; BD Pharmingen, 553,035) on ice at 4 °C in the dark. CD3⁺ CD8⁺ T cells in the lower compartment were counted using Precision count beads (BioLegend, 424,902).