Hepes buffer ph 7.2

THP-1 cells were obtained from American Type Culture Collection (ATCC) and maintained in complete R10 media [RPMI-1640 (Gibco, cat#: 11875119) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, cat#: 12007C), 5% penicillin/streptomycin (50μg/ml; Gibco, cat#: 15140163), 5% l-glutamine (4 mM; Gibco, cat#: A2916801), and 5% Hepes buffer (pH 7.2) (50 mM; Gibco, cat#: 15630106)]. Cell culture densities were kept below 0.5 × 10⁶ cells/mL to maintain consistent assay performance.
To generate peripheral blood mononuclear cells (PBMCs), whole human blood with ethylenediamine tetraacetic acid (EDTA) was mixed at a 1:10 ratio with ammonium-chloride-potassium (ACK) lysis buffer (Gibco, cat#: A1049201) and incubated for 10 min at room temperature. The PBMCs were pelleted by centrifugation (500 ×g, 5 min) at room temperature and then washed with cold PBS. The WBCs were finally diluted in complete R10 media for ADNP assay.

Human embryonic kidney epithelial cells (HEK293; ATCC, CRL-1573; HEK293T, ATCC, CRL-11268) and Human bone osteosarcoma epithelial cells (U2OS; ATCC, HTB-96) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, 11965092) supplemented with 10% (v:v) heat-inactivated fetal bovine serum (FBS; Gibco, 10100147), 1% (v:v) penicillin–streptomycin (Gibco, 15140148), 1 mM sodium pyruvate (Gibco, 11360070), 2 mM L-glutamine, 10 mM HEPES buffer (pH 7.2, Gibco, 15630106), 50 µM 2-mercaptoethanol (Gibco, 21985023). Cells were maintained at 37°C in 5% CO₂. All cells included in this research were free of mycoplasma confirmed by the LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich, MP0035).