Coculture of Salivary Epithelial Cells and PBMCs

Human peripheral blood mononuclear cells (PBMCs) were purchased from Lonza. Healthy human salivary gland epithelial cells (SGECs) were isolated and cultured as we reported previously [24 (link)]. The SGEC-PBMC coculture experiment was based on a published protocol [25 (link)]. Briefly, SGECs were seeded at 1?×?10⁵ cells per well into 12-well plates and cultured in Keratinocyte serum-free medium (SFM, Life technology) with poly I:C (5?μg/ml, InvivoGen, tlrl-picw) for 12 hours to allow attachment, stimulation of autoantigen synthesis, and IL7 expression essential for SS progression. After removing the SFM and washing with PBS, 2?×?10⁴ PBMCs per well were added in LGM-3 lymphocyte growth medium (Lonza) containing 10% FBS and phytohemagglutinin-P (PHA-P, 5?μg/ml, Sigma-Aldrich, L8754) to activate T cells. After 4 days of coculture, SGECs and PBMCs were harvested together and analyzed for gene expression by qRT-PCR.