Elisa maxtm standard set

Culture supernatants obtained from the B16–BL6/Zs green proliferation assays (see above) at 24 and 96 h of incubation with 1000 or 2000 μg/mL of gabapentin (most effective doses) were studied (Figure 9). Moreover, we included supernatants from culture plates after gabapentin was washed out, which were maintained with fresh culture medium for an additional 72-h period, as well as control supernatants from plates without gabapentin. A commercially sandwich enzyme-linked immunosorbent assay kit (ELISA Max^TM Standard Sets, BioLegend, San Diego, CA, USA) was used to determine the chemokine CCL2 protein concentration (pg/mL) on enzyme-linked immunosorbent assay microplates (MaxiSorp, Nunc^TM, Apogent, Portsmouth, NH, USA). CCL2 concentration was estimated based on an appropriate internal standard curve using a recombinant murine chemokine. The results represent the mean of three independent assays, conducted in triplicate and expressed as the mean ± SEM.

Cytokine concentrations in the samples were determined using ELISA MAX^TM Standard Sets (BioLegend) according to the manufacturer’s instructions. The data are expressed as mean ± standard deviation for the samples, which were assayed in triplicate. At least 3 independent experiments were conducted.

Human T cells were co-cultured with or without K562, K562/CD19, or Raji cells, respectively, in the presence of the OVV or OVV-CD19BiTE supernatants (100 μl) or blinatumomab (100 ng/ml) for 24 h. Then supernatants were obtained from this co-culture system. Samples were centrifuged at 600 × g for 5 min at room temperature and the supernatants were collected. Human IFN-ɣ, TNF-α, and IL-2 in the supernatants were quantified using the ELISA MAXTM Standard Set (Cat#430115, 430216, 431815, Biolegend, USA) according to the manufacturing protocol.

The amount of IL‐5, IL‐13 or IFN‐γ in BALF or culture medium was determined according to the protocol of by ELISA MAX^TM standard set (Biolegend) or DuoSet ELISA kit (R&D systems) with slight modification. ELISA 96‐well half plates (Corning, NY, USA) were coated with anticytokine antibody (IFN‐γ (BioLegend), IL‐5 (BioLegend) and IL‐13 (R&D systems)) in 0.1 N carbonate buffer at 4 °C overnight. After washing three times with PBS containing 0.05% Tween 20 (PBS‐T), the wells were blocked with 1% BSA in PBS for 1 h at room temperature. Samples and standards were added. Nonstimulated samples with OVA were included as a negative control. After incubation at room temperature for 2 h, the wells were washed three times with PBS‐T. Biotin‐conjugated anti‐cytokine antibody was used as detection antibody. After incubation at room temperature for 1 h, the plates were extensively washed with PBS‐T. Streptavidin–horseradish peroxidase (HRP) was added. After washing three times with PBS‐T, specific cytokine binding was visualised by adding TMB solutions, and then, the reaction was terminated by 1 N H₂SO₄. Finally, the absorbance at 450 nm was measured by microplate reader (Epoch, BioTeK, CA, USA).