High-throughput assays for gingival fibroblast in vitro wounds treated with nicotine, and/or TGF1
Conference Paper
Overview
Research
Identity
Additional Document Info
View All
Overview
abstract
Nicotine treated gingival fibroblast cells have inhibited wound and TGF1 responses leading to altered phosphorylation of signaling proteins. Over 600 signaling proteins were screened for changes in phosphorylation when treated with nicotine and/or TGF1 and wounded in vitro and a highthroughput cell culture assay was developed to screen effects on wound closure. Primary human fibroblasts were grown to confluence in 30mm dishes and 96 well plates, starved for 24 hours and treated with nicotine or TGF1 2 hours before wounding. Four treatment groups: controls (C), nicotine (N), TGF1 (TGF), and N+TGF were divided into wounded and unwounded groups. Cells were lysed in ice cold buffer with phosphatase inhibitors, frozen and transported to Kinexus for phosphorylated protein screening. A 96 well plate pin array was used to uniformly wound each well. Plates were fixed and stained at 0, 4, 7, 14, and 24 hours. Plates were imaged with a light scanner at 1200 dpi. Wound areas were measured with Metamorph. Proteins involved in cell migration including p38 MAP kinase, MEK1, and PCTK1 were increased in C and TGF, but decreased in N and TGF treated groups. N treatment increased phosphorylation of CDK1/2 and PKCd. The pin array wounded the cells in a uniform and predictable manner. Wounds had closed 5056% by 4hr. Nicotine treated wounds were larger at 7 and 14 hr and closed at 24 hr.
