Streptomycin sulfate

CII-specific T cells from draining LNs were enumerated and characterized using a DR1-CII tetramer [28 (link)]. Briefly, soluble DR1 covalently linked to the immunodominant CII peptide was produced in S2 cells, affinity-purified, biotinylated by BirA (Avidity, Aurora, CO, USA), and formed into multimeric units by stepwise addition of PE-labeled streptavidin (Rockland Immunochemicals, Limerick, PA, USA). For identification of CII-specific T cells ex vivo, LN cells were incubated with 1 μg of the tetramer at 37 °C for 2.5 h in complete HL-1 medium (50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate, 0.05 mM β-mercaptoethanol, 2 mM l-glutamine, and 0.1 % bovine serum albumin; BioWhittaker/Lonza, Walkersville, MD, USA) supplemented with 5 mM NaN₃. At the end of the incubation, antibodies specific for various CD markers were added and cells were incubated for an additional 30 minutes at 4 °C. Samples were then washed and resuspended in PBS supplemented with 0.1 % NaN₃ and 2 % fetal bovine serum and analyzed by flow cytometry (BD LSR II). DR1-restricted, CII_257–274-specific and HA_306–318-specific T-cell hybridomas were used as positive and negative controls, respectively, to monitor the specificity and sensitivity of the DR1-CII tetramer (see Additional file 1).

Jurkat and MOLM-13 cells were purchased from the DSMZ (Braunschweig, Germany). Cells were cultured in RPMI 1640 medium with stable l-glutamine (Lonza, Cologne, Germany) supplemented with 10% (Jurkat) or 20% (MOLM-13) FCS (Capricorn Scientific, Ebsdorfergrund, Germany), 100 units/ml penicillin G sodium salt, and 100 µg/ml streptomycin sulfate (Lonza). Cells were maintained in a humidified atmosphere at 37 °C and 5% CO₂. Cells were tested to be negative for mycoplasma with the qPCR Mycoplasma Test Kit from Applichem (Darmstadt, Germany).

Murine macrophage cell line RAW 264.7 cells, obtained from American Type culture collection, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 8% fetal bovine serum (FBS) (WelGene Co., Daejeon) and 100 IU/mL penicillin and 100 μg/mL streptomycin sulfate (Lonza, MD, USA). The incubating conditions were maintained at humidified 5% CO₂ at 37°C.

The human keratinocyte cell line, HaCaT, was purchased from CLS (#300493, Cell Lines Service, Eppelheim, Germany). Normal human dermal fibroblasts, neonatal (NHDF), were purchased from Thermo Fisher Scientific (#C-004-5C, Waltham, MA, USA). HaCaT cells and NHDF were cultured in Dulbecco’s modified Eagle’s medium (DMEM; #12-604F, Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum (#10082-147, Thermo Fisher Scientific), 100 U/mL potassium penicillin and 100 mg/mL streptomycin sulfate (#17-602E, Lonza) at 37 °C in a humidified 5% CO₂ incubator. The rate of cell viability was quantified using the Cell Counting Kit-8 (CCK-8) reagent (#CK04-11, DOJINDO, Tokyo, Japan) according to the manufacturer’s instructions.