Mouse il 2 elisa set

iBMDM cells were seeded in 24-well plates. After the cells became attached and reached 50% confluence, they were transfected with 500 ng of GFP, Nef(1 to 115)-GFP-Flag, or Nef(1 to 115)-(L112A/Y115A)-GFP-Flag plasmids for 15 h. After cells were washed 3 times with PBS, Opti-MEM I reduced serum medium (Thermo Fisher) supplemented with 10 mg/ml OVA was added for 6 h. The cells were then washed 3 times with PBS and cocultured with B3Z cells for 24 h. The medium was collected, centrifuged at 12,000 rpm for 10 min to discard the cell debris, and frozen at –80°C until analysis. The IL-2 concentration was measured using the mouse IL-2 ELISA set (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol.

The suspension of spleen cells from immunized mice was prepared by mechanical disruption. Ammonium chloride lysing buffer was used for the lysis of red blood cells. The prepared cells were seeded into 96-well cell culture plates at a density of 1 × 10⁶ cells/mL and stimulated either with VLPs (HaVP1-PADRE-4, TSPyV VP1 and HaPyV VP1) at a concentration 5 μg/mL or polyclonal activators Concavalin A (ConA) (Sigma-Aldrich) and lipopolysaccharide (LPS) (Sigma-Aldrich) added at concentrations 3 μg/mL and 1 μg/mL, respectively, to the growth medium: RPMI-1640 containing 10% FBS (Merck-Millipore) and antibiotics. After 24 h and 48 h of incubation, culture supernatants were collected and stored at −80 °C until analysis.
Interleukin 12 (IL-12), interleukin 2 (IL-2) and interferon gamma (IFN-γ) concentrations in mouse spleen cell cultures were determined by sandwich ELISA using Mouse IL-12/IL-23 total p40 ELISA Ready-Set-Go, Mouse IFN gamma ELISA Ready-Set-Go (eBioscience, San Diego, CA, USA) and Mouse IL-2 ELISA set (BD Biosciences, San Jose, CA, USA) according to the recommendations of the suppliers.