α cd8 microbeads

In vitro generated Th1 or Th17 cells were transferred i.v. into RAG KO mice, along with polyclonal CD8⁺ T cells purified from naïve BALB/c mice by positive selection using α-CD8 microbeads (Miltenyi Biotec, Auburn, CA). RAG KO mice received 0.5–2 x 10⁶ Th1 or Th17 cells, and 3–5 x 10⁶ CD8 T cells, by tail vein injection and were challenged s.c. one day later with 5,000 CMT. Parasitemia was measured post-infection microscopically using 1.5 μl peripheral blood taken from the tip of the tail. Protection was also assessed by survival of infected mice. At 3, 6, 7 and 10 days post-infection, the spleens from representative mice were harvested for immune studies.

LSEC were isolated as described previously [36 (link)] and purified by immunogenic separation using αCD146-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), achieving purity of >98% and plated into collagen-coated 24-well or 96-well plates. One day after isolation, cells were washed with PBS containing 1% Fetal Calf Serum (FCS) and used two days later for experiments. Kupffer cells were isolated after perfusion of the liver with a collagenase solution and subsequent digestion with collagenase in vitro. After a 50%/25% Percoll gradient (GE Health Care, Freiburg, Germany), cells were plated, incubated at 37 °C and washed after 30 min. Adhering Kupffer cells were then used for experiments. After LPS stimulation of LSEC and Kupffer cells, supernatants were harvested at 24 and 48 h and IL-6 content was measured by ELISA. Dendritic cells (DC) were purified by immunogenic separation using αCD11c-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) after collagenase-digestion of spleens according to the manufacturer’s instructions. CD8+ T cells were isolated from spleens and lymph nodes of H2-Kb-SIINFEKL-restricted OT-I TCR-transgenic mice by αCD8-microbeads (Miltenyi Biotec).