Cd43 microbeads beads

Serum and spleens were harvested from C57BL/6 wt, IDO1 ko, IDO2 ko, or dko mice Spleens were pooled from 3 mice/genotype prior to purification. T cells were purified by MACS bead magnetic purification (Miltenyi Biotec) using CD90.2 microbeads for total T cell purification by positive selection and B cells were purified using CD43 microbeads beads (Miltenyi) by negative selection. T cell purity was >93% and B cell purity >96%. Cells were cultured for 48 h in media alone (unstimulated), 5 μg/mL plate-bound αCD3 + 2 μg/ml soluble αCD28 (T cells), or 25 μg/ml LPS (Sigma-Aldrich) + 50 ng/mL IL-4 (B cells). Human 293-T-REx™ cells stably transfected with murine IDO1, IDO2, or untransfected controls (parental) under the control of the Tet repressor were used as positive controls for enzyme activity. IDO1 and IDO2 expression in 293-T-REx™ cells was induced with 2 μg/ml doxycycline for 48 h. Serum and harvested supernatants were analyzed for kynurenine levels using the IDK high sensitivity Kynurenine ELISA kit according to manufacturers instructions (Immunodiagnostik). Kynurenine levels were calculated by comparison to a standard curve.

Liver and epididymis tissue were harvested from individual wt, IDO1 ko, IDO2 ko, and dko C57BL/6 mice. Spleens from C57BL/6 wt, IDO1 ko, IDO2 ko, or dko mice were harvested from 3 mice/genotype and pooled prior to purification. T cells were purified by MACS bead magnetic purification (Miltenyi Biotec) using CD90.2 microbeads for total T cell purification by positive selection and B cells were purified using CD43 microbeads beads (Miltenyi) by negative selection. T cell purity was >93% and B cell purity >96%. Cells were cultured in media alone (unstimulated), 5 μg/mL plate-bound αCD3 + 2 μg/ml soluble αCD28 (T cells), or 25 μg/ml LPS (Sigma-Aldrich) + 50 ng/mL IL-4 (B cells). After 48 h, cells were harvested, RNA was extracted with the RNEasy mini kit (Qiagen), and first strand cDNA synthesized using oligo-dT primer (Promega GoScript). IDO1 and IDO2 expression were measured by real time PCR using SYBR Green (Sigma-Aldrich). Expression of target gene IDO2 was determined relative to β-2-microglobulin (β2M) and calculated as 2∧⁻(Ct_Targetgene-Ct_b2M) as primers had similar efficiencies. Primers: IDO1, 5′-CCCACACTGAGCACGGACGG-3′ and 5′-TTGCGGGGCAGCACCTTTCG-3′, IDO2, 5′-CAATCCAGCCATGCCTGTGGGG-3′ and 5′-TGGGCTGCACTTCCTCCAGAGT-3′, and β2M 5′-CTCGGTGACCCTGGTCTTTC-3′ and 5′-TTGAGGGGTTTTCTGGATAGCA-3′.