Human il 10

Cell culture supernatants were obtained and centrifuged to spin down the cells. Cell-free supernatants were used in sandwich ELISAs as instructed by the producers of the antibody pairs used. Samples were analyzed in duplicates for murine TNF, murine IL-6 (R&D Systems), murine IL-1β, murine IL-12 (eBiosciences), murine IL-17 (eBioscience), human IFN-γ and human IL-10 (R&D Systems). Lower limit of detection was 30 pg/ml, whereas upper limit of detection was 10 ng/ml for all of the ELISA tests performed. Samples that showed values over the upper limit of detection were adequately diluted for the measurement. The results were calculated using standard curves made on the basis of known concentrations of the appropriate recombinant cytokines.

Arg1 expression and arginase enzymatic activity in BMDM was determined at 24 hrs or 48 hrs by real-time RT-PCR or by production of urea, respectively, as described previously [2] (link). For Western blot a goat anti-hamster arg1 polyclonal antibody was used [2] (link). The antibody used for detection of hamster arg1 did not react with L. donovani parasite lysates. The cells were left unstimulated or exposed to recombinant human Epidermal Growth Factor (EGF), mouse Insulin-like Growth factor-1 (IGF-1), human Platelet-Derived Growth Factor (PDGF) (Cell Signaling), human Fibroblast Growth Factor basic (heparin stabilized) (Sigma), 0.3–2.5% recombinant hamster IL-4 conditioned medium (equivalent to 3–25 IU/mL determined by STAT6 reporter bioassay) [2] (link), or human IL-10 (R&D Systems) and/or infected with L. donovani promastigotes at 1∶2 macrophage∶parasite ratio. The activity of human IL-10 on hamster cells was verified using hamster BMDMs transiently transfected with a STAT3 lentiviral reporter construct (Cignal Lenti-reporter, SA Biosciences).

MDA-MB-231 human breast adenocarcinoma cells (American Type Culture Collection, Manassas, VA) were cultured in L-15 Leibovitz’s Medium (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA) at 37 °C with atmospheric CO₂. The human foreskin fibroblast (HFF) cell line (ATCC) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS at 37 °C in a humidified chamber with 5% CO₂. Human cytomegalovirus strain AD169 (ATCC) was used to infect confluent monolayers of HFFs at the indicated multiplicities of infection. Purified recombinant cmvIL-10, human IL-10, and epidermal growth factor (EGF) were purchased from R&D Systems (Minneapolis, MN). IL-10R neutralizing antibody and S3I-201 Stat3 inhibitor were from Santa Cruz Biotechnology (Santa Cruz, CA).