Mouse ifn γ elisa kit

For serum biochemistry, 1 mL of whole blood was collected from vena cava at sacrifice under Zoletile mixture (Zoletile 50; Virbac Lab., Paris, France) anesthesia and separated the serum. All serum samples were frozen at −150°C until they were assayed. Serum IL-6 levels werosteocalcin levels were detected by enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA) as pg/mL according to previously established method [30 (link)], and serum IFN-γ levels were also calculated using mouse IFN-γ ELISA kit (BD Biosciences/Pharmingen, San Diego, CA, USA) according to manufacturer's recommended protocols as pg/mL levels.

The IFN-γ concentration in mouse serum was measured using a mouse IFN-γ ELISA kit (BD Biosciences) according to the manufacturer's protocol. The IL-2 concentrations in the cocultures of T cells and APCs were measured using the following Abs: anti-IL-2 (JES6-1A12) for capture, biotinylated anti-IL-2 (JES6-5H4) and streptavidin-HRP for detection (all from BD Biosciences).

To show that doxorubicin has the ability to induce immunogenic cell death in neuroblastoma cells, doxorubicin or CDDP-treated neuro-2a cells were co-cultured with BM-DCs and CD8α⁺ lymphocytes, and IFN-γ concentrations in the culture supernatants were compared. CDDP was used as a control agent as it is unable to induce immunogenic cell death in tumor cells (14 (link)). Neuro-2a cells (2.0×10⁵) treated with either doxorubicin (5 μM for 24 h) or CDDP (25 μg/ml for 72 h) were co-cultured with CFSE-labeled CD8α⁺ cells (2.0×10⁵) in 24-well plates coated with hamster anti-mouse CD3/CD28 antibodies (BD Pharmingen). Following three days of co-culture, CD8α⁺ cell proliferation was confirmed using FACS, and IFN-γ concentrations in the culture supernatants were measured using a mouse IFN-γ ELISA kit (BD Biosciences).

Retro-orbital blood was obtained via EDTA-coated micro capillary tubes during organ harvest at the end of the study. The whole blood of mice after immunization had been analyzed by Celltac F automated hematology analyzer to investigate the blood components, including leukocytes, erythrocytes, thrombocytes, lymphocytes, monocytes, and neutrophils. The remaining whole blood was spun at 10,000 rpm for 10 min at 4°C. The supernatant was collected and frozen at −20°C until analysis to reduce multiple freeze/thaw cycles. No-fasting plasma lipids, including total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides levels, were determined using plasma biochemistry automatic analyzer (Hitachi High-Technologies Corp., Minato-ku, Tokyo, Japan) as described previously (31 (link), 32 (link)). Mouse IFN-γ ELISA Kit, Mouse IL-6 ELISA Kit, and Mouse MCP-1 ELISA Set were purchased from BD Pharmingen. Mouse TGF-β1 Precoated ELISA kit were provided by Boster Biological Technology Co., Ltd. The levels of IFN-γ, IL-6, MCP-1, and TGF-β1 were determined according to the manufacturer's protocol after the plasma samples being diluted at 1:2.

The production of IL-4 and IFN-γ cytokines was quantified using ELISA (Mouse IL-4 ELISA kit and Mouse IFN-γ ELISA kit- BD OptEIA) according to the manufacturer’s protocol (BD Biosciences, San Diego, CA, USA). Cell suspensions were prepared from the lymph nodes and spleens of BALB/c mice infected for 4 weeks with LbrC¹, LbrM¹, LbrC² and LbrM² promastigotes. Overall, 5x10⁶ cells/mL were plated per well in 24-well tissue culture plates and stimulated with 40 μg/mL L. braziliensis particulate antigens (SLA) as previously described [22 (link)].