Quantitation of Rat Lacrimal Secretion: a Novel Sandwich ELISA with High Sensitivity

cDNA and genomic cloning of lacritin, a novel secretion enhancing factor from the human lacrimal gland11Edited by J. Karn

“…For analytical comparison, small amounts of mammalian lacritin were expressed in 293T cells using pcDNA3.1/myc-His() (Invitrogen, Carlsbad, CA) containing lacritin insert, and then puri®ed under native conditions. Preimmune serum and anti-bacterial lacritin antiserum were subsequently prepared in rabbits (Covance Research Products, Denver, PA), partially puri®ed by precipitation in 4 % caprylic acid 67 and treatment with 293T cell acetone extract (to eliminate background), and assessed by ELISA (1/1000 dilution) using recombinant bacterial lacritin (4 mg/ml) as coat. For immunohistochemistry, sections of zinc formalin-®xed, paraf®n-embedded human tissues and a human tissue microarray were deparaf®nized and rehydrated, and microwave heated (20 minutes in 10 mM citrate buffer (pH 6.0)) to expose antigen.…”

“…Freshly isolated rat lacrimal acinar cells 20,47,67 and HSG (human salivary gland) ductal 23 and HCE (human corneal epithelial) 68 cell lines were used to study lacritin function. For secretion studies, rat acinar cells were plated serum-free overnight on wells co-coated with 0.05 mM laminin-1 (to ensure adhesion) and 0 to 20 mM lacritin, or alternatively with laminin-1 (0.05 mM) and treated the next day with serum-free medium containing 0 to 162 ng/ml of soluble lacritin for four hours.…”

Use of nucleofection to efficiently transfect primary rabbit lacrimal gland acinar cells

“…Many studies investigating the mechanisms associated with the development of dry eye disorders and the autoimmune disease, SjS, utilize rabbit LGAC cultures (Jerdeva et al 2005;Marchelletta et al 2008;Mircheff et al 1998;Xie et al 2002) while others use rat and mouse LGAC (Chen et al 1998;da Costa et al 2006;Hodges et al 2004;Rios et al 2005;Sanghi et al 2000;Zoukhri and Kublin 2002). Primary cultured LGAC have previously exhibited very low transfection efficiency using non-viral, lipid-based methods, thus significantly limiting the types of experiments that may be conducted.…”