Pan t cell beads

Single cell suspensions of spleens were prepared in PBS + 1% FBS by grinding with a sterile syringe plunger and dispersed by pipetting, then filtering through a 40 μ cell strainer. For co-culture experiments, splenic OT-I or WT CD8 T-cells were prepared by first enriching for T-cells using Pan-T-cell beads and further purified by negative selection using appropriate magnetic beads (Miltenyi). All bone marrow and splenic T-cells purified by positive selection were incubated for 30 m at 37° C to allow cells to allow dissociation or uptake of bound beads from cell surface. The resulting T-cells were routinely > 97% pure when stained with anti-CD3, anti-CD4 and anti-CD8 antibody.

Splenocytes were isolated as previously described (18 (link)). Briefly, single cell suspensions of spleens were prepared in PBS+1% FBS and filtered through 40μ cell strainer. CD8 T-cells were prepared by first enriching for T-cells using Pan-T-cell beads then purified by negative selection using appropriate magnetic beads (Miltenyi; Auburn, CA). To generate Tc_REG, day 3 osteoclasts were plated at 5×10⁵ cells/well in 24-well tissue culture plates (Corning Inc., Corning, NY, USA). Next day, osteoclasts were pulsed with a peptide derived from residues 257–264 (SIINFEKL) of ovalbumin (5 μM; AnaSpec Inc.) for 3 hours. Freshly isolated, 2.5×10⁵ cells/well OT-I splenic CD8 T-cells were then added in 2 ml of RPMI supplemented with 10% heat inactivated FBS, penicillin-streptomycin-glutamine, non-essential amino acids, 10 mM HEPES, and 55 μM β-mercaptoethanol. M-CSF and RANKL were added at 20 ng/ml and 100 ng/ml, respectively. For polyclonal CD8 T-cells from C57BL/6 mice α-CD3 antibody at 1 μg/ml was used instead of OVA peptide and was added to the T-cells before plating them on osteoclasts. In some experiments γ-secretase inhibitor DAPT and IKK inhibitor IKK-16 (both from Selleckchem) were added to the media at 10 μM concentration.