Ab91356

Blood was collected at selected time points posttransplantation through retro-orbital bleeding and collected in BD vacutainers. Briefly, lymphocytes were isolated by the Histopaque gradient separation method (52 (link)). After centrifugation at 400 × g for 30 min, the buffy coat of lymphocytes was aspirated, and specific Treg markers (CD4, CD25, and FOXP3) were stained with fluorescently labeled rat anti-mouse antibodies, i.e., APC-CD4⁺ (Clone RM4-5 RUO), PE-Cy7 CD25⁺ (Clone PC 61 RUO), and PE-conjugated FOXP3⁺ (Clone MF23 RUO), respectively, as recommended by BD Pharmingen FOXP3 fixation and permeabilization assay, which specifically flow sort the CD4⁺CD25⁺FOXP3⁺ Treg subpopulation from mixed lymphocytes in systemic circulation. All were referenced to isotype control mouse IgG1-fluorescein isothiocyanate (FITC) (ab91356, abcam) to determine the background due to nonspecific antibody binding, and T cells were confirmed with CD4 and CD3 coexpression. Data were recorded at the flow rate of 14 μl/min and a minimum of 500,000 events were collected, and further analyzed through BD Accuri integrated software version C6.

The isolated cells were identified by flow cytometry assay with lineage-specific markers, including anti-CD90 (ab33694, Abcam, UK) and anti-CD31 (ab33858; Abcam, UK). Isotype controls (ab91357, ab91356; Abcam, UK) were used for anti-CD90 and anti-CD31, respectively. TDSCs (5 × 10⁵) at P3 were incubated with fluorescein-conjugated anti-rat monoclonal antibodies for 1 h at 4°C and centrifuged at 1000 rpm for 5 min. The stained cells were resuspended in 500 μL ice-cold PBS containing 10% FBS and analyzed by FACs (FACSCalibur, Becton Dickinson) [23 (link)].

Non-small cell lung cancer (A549, NCI-H292, NCI-H1299 and NCI-H358), hepatocellular cancer (SNU449, HuH1, HuH7, Chang, Hep3B, HepG2), colon cancer (WDr) and breast cancer (MCF-7) cell lines, obtained from American Type Culture Collection (ATCC), were cultured in complete RPMI 1640 medium (Gibco) supplemented with 10% FBS (Thermo Scientific) and 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Polyinosinic-polycytidilic acid (polyI:C), is a synthetic short-chain analog of dsRNA from InvivoGen. Actinomycin D, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Stattic were from Sigma. Tyrphostin AG 490 was from Calbiochem. Antibodies used were rabbit anti-IL6 polyclonal antibody (ab6672, Abcam), ChromPure rabbit and mouse IgG control, whole molecule (Jackson ImmunoReseach), functional grade purified anti-human TLR3 (e-Bioscience), mouse IgG1 (ICIG1) (FITC) isotype (ab91356, Abcam), anti-TLR3 antibody (40C1285.6) (FITC) (ab45053, Abcam), anti-STAT3 (phospho Y705) antibody and anti-STAT3 antibody (Abcam), anti-JAK2 and Phospho-JAK2 (Cell Signalling), Caspase-3 (8G10, Cell Signalling).