6.

Extracellular calcium is essential for nanopore-induced signaling. (a–c) Primary human T cells were seeded and incubated for 30 min on the indicated substrates in (a, b) T cell medium containing EDTA (2 mM) or EGTA (1 mM) with additional magnesium (Mg2+, 0.6 mM) or in (c) HBSS medium containing the indicated concentration of calcium (Ca2+) or Mg2+. (a, c) Median fluorescent intensity (MFI) of phosphorylated ERK measured by flow cytometry, normalized per donor to all positive control samples. (a): Flat + aCD3/28 and nanopores; (c): flat+aCD3/28 ± 1.6 mM Ca2+ and nanopores (+1.6 mM Ca2+) and naive as baseline. (b) NFAT MFI in isolated nuclei measured by flow cytometry, normalized per donor to the positive control sample Flat+aCD3/28 and naive as baseline. (a, b) Bars indicate the mean ± SD across donors (D, (a): n _D = 3–4; (b): n _D = 4–6). Symbols indicate mean values of technical replicates for each donor. (c) Points indicate the mean ± SD across donors (nD = 3). (a–c) Statistical analysis was performed on per-donor mean values from donors present in both conditions using a paired t test with Benjamini–Hochberg (FDR) adjustment for multiple comparisons. Significance symbols represent adjusted p-values: ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, and ***: p < 0.001.