Figure 3.

Quantification of IFN-γ in single CD8 T cells. (A) Reproducibility of the SiMoA standard curves for IFN-γ. Standard concentrations of IFN-γ were used to generate curves to calculate the unknown concentrations in cells (B) Comparison of measured IFN-γ protein at 1, 2, 5, and10 cell levels in CD8 cells. The amount of IFN-γ measured in femtograms of lysates from cell surface IFN-γ-positive cells or cells that were cell surface IFN-γ-negative. Left are data from IFN-γ-positive cells, right are data from IFN-γ-negative cells. Horizontal axis is the number of cells sorted into each lysate. (C) Competitive inhibition assay for IFN-γ assay. Five and ten cells were sorted in 25 μl of lysis buffer and the lysates were incubated with or without anti-IFN-γ inhibition antibody for 45 mins at 4°C. Lysates were transferred to V bottom SiMoA plates and IFN-γ assay was performed. Data are represented as mean and mean ± SD and differences among means were calculated using Mann–Whitney test. P < 0.05 were considered significant.