Hi fcs

Peripheral blood mononuclear cells (PBMCs) were separated by density gradient over Ficoll-Paque (GE Healthcare) as previously described (36 (link)). PBMCs from patients with AP and AAH and corresponding controls were cryopreserved in heat inactivated fetal bovine serum (HI-FCS [ThermoFisher Scientific]), 10% dimethyl sulfide (DMSO [Santa Cruz Biotechnology]). All other experiments used freshly isolated PBMCs with no cryopreservation. Viability of all PBMCs were assessed prior to all experiments using trypan blue (Sigma Aldrich) and only those with viability over 70% for cryopreserved samples and 90% for fresh samples, were included. Cells were seeded at 1 million per mL in RPMI 1640 containing L-glutamine (Lonza), penicillin/streptomycin (Sigma Aldrich), and 10% HI-FCS. PBMCs were stimulated with 20 ng/mL lipopolysaccharide from Escherichia coli O55:B5 (LPS [Sigma-Aldrich]), 10 μg/mL polyinosilic:polycytidylic acid (poly I:C [Sigma-Aldrich]), 100 ng/mL flagellin from Salmonella typhimurium (Source BioScience), 1 μg/mL high mobility group box 1 protein (HMGB1 [Sigma-Aldrich]), 1,000 units/mL interferon alpha (IFNα [Peprotech]) or 50 ng/mL interleukin 1 alpha (IL-1α [R&D Systems]) for 3 or 24 h in 12 well plates, with 1 mL per well. Monocytes were magnetically isolated using the Pan monocyte isolation kit (Miltenyi Biotec) according to the manufacturer's instructions.

L. major MHOM/SA85/JISH118 and L. mexicana MNYC/BZ/62/M379 parasites were cultured in Schneider's insect medium (Sigma, UK) supplemented with 10% heat-inactivated fetal calf serum (HiFCS; Sigma UK). These were passaged each week at a 1:10 ratio of existing culture to fresh media in 25-ml culture flasks without a filter and incubated at 26°C. For infection of mice, stationary-phase parasites were centrifuged for 10 min at 2,100 rpm and 4°C. The supernatant was removed and the pellet resuspended in RPMI medium (Sigma, UK). Cell number was estimated by microscopic counting with a Neubauer hemocytometer. AmBisome (LAmB; Gilead, UK) was reconstituted with 12 ml sterile water (as per the manufacturer's instructions) to yield a stock solution of 4 mg/ml and diluted in 5% aqueous dextrose to achieve a drug dose of 25 mg/kg. Paromomycin sulfate (Sigma) was prepared in phosphate-buffered saline (PBS) to yield 50-mg/kg doses. Lambda carrageenan (Sigma) and Evans blue (Sigma) 0.5% (wt/vol) solutions were made up in phosphate-buffered saline (PBS; Sigma). The drug preparations were stored at 4°C during the experiments.

Where indicated cells were re-suspended in RPMI-1640, 10% heat-inactivated fetal calf serum (HIFCS; Sigma-Aldrich) and stimulated with phorbol myristate acetate (PMA; 50 ng/ml), ionomycin (750 ng/ml) and Brefeldin A (2 mg/ml) (all Sigma-Aldrich) for 3 h at 37 °C, 5% CO₂. This protocol has been widely used to reveal the cytokine production capabilities of T cells, including studies on MS (Mazzoni et al., 2015 (link)). Cells were incubated with antibodies specific for surface markers for 20 min at 4 °C before washing. To detect CCR6, cells were stained at room temperature before and after cell culture. Where required, cells were fixed and permeabilised according to the manufacturer’s instructions (FIX & PERM®, Life Technologies) before staining with antibodies specific to intracellular markers. Cells were re-suspended in phosphate-buffered saline (PBS) 2% bovine serum albumin (BSA) for flow cytometric analysis, which was performed using a CyAn™ ADP flow cytometer with data analysed using Kaluza® Flow Cytometry Analysis Software (Beckman Coulter). Isotype control antibodies or unstimulated controls (for cytokine analysis) were used to determine positivity.

293T/17 [HEK 293T/17] cells were obtained from the American Type Culture Collection (ATCC CRL-11268). TZM-bl dual-reporter cell line was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl ARP-8129, contributed by Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% V/V HIFCS, 10 mM Hepes, 100 U/mL penicillin and streptomycin (all Sigma, UK) at 37°C ± 5% CO₂.

Homogenous macrophage populations were generated by isolating peripheral blood mononuclear cells cells (PBMC) from the venous blood of healthy volunteers incubating pellets for at least 15 minutes in 20 µl of CD14⁺ beads (Miltenyi Biotech, UK) and 80 µl of MACS buffer (Miltenyi Biotech) per 1×10⁷/ml PBMC. Cells were then washed twice using MACS buffer and passed through a MACS column inserted into a magnetic holder. Negative subsets were excluded in the run through of column. The column was then removed from the holder and CD14⁺ cells were collected by washing and set up in complete media at an appropriate concentration. To grow M1 or M2 macrophages either 10 ng/ml of recombinant human GM-CSF (PeproTech, UK) or 50 ng/ml recombinant human M-CSF (PeproTech, UK), respectively, was added to purified monocytes in complete medium; RPMI 1640 media supplemented with 10 mM L-Glutamine, and 5000 U/ml penicillin with 10 mg/ml Streptomycin (GPS) with 10% heated inactivated fetal calf serum (HIFCS) (all Sigma UK) and 4% human serum (HD Supplies UK) and incubated at 37°C in a humidified atmosphere of 5% CO2 for 6–7 days and harvested for use.

HEK293T cells grown in DMEM media supplemented with 10% Ig-depleted HI-FCS (Sigma), 1 mM Glutamax, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) were transiently transfected with pADGRE1-pFUSE-hIgG1-Fc2 DNA at a ratio of 1:1 with Lipofectamine 2000 (Life Technologies) following manufacturer's instructions. Four days post-transfection, the supernatant was harvested, centrifuged to remove debris and filter sterilized. Soluble protein was then purified using a HiTrap Protein G column (GE Healthcare) and desalted using Slide-A-Lyzer™ G2 Dialysis Cassettes 1–3 mL 10 K MWCO (Life Technologies). The recombinant protein was used as an immunogen for monoclonal antibody (mAb) production.