Mast cell culture and sphingolipid receptor expression on IgE
exposure:
Mast cell lines were purchased from (Kerafast-LUVA-Human Mast Cell Line,
(EG1701-FP, MA, USA). Suspended cells were grown to confluence in six
well culture plate and were exposed to 10ng/ml & 20ng/ml concentration
of IgE (Abcam-Native human IgE protein (ab65866, MA, USA) for 16
hours in serum free medium (STEM PRO-34 nutrient supplement (Gibco
#10641-025, MA, USA); Pen Strep (Gibco #15140, MA, USA);
L-glutamine-200 mM (Gibco # 25030-081). At the end of 16 hours, cells
were centrifuged at 3000g, washed in 1X-PBS. RNA was isolated from
these cells using Trizol (FavorPrep™ Tri-RNA Reagent, Merck, St.Louis,
USA) followed by cDNA conversion (Biorad-iScript 1708841, CA, USA). The
gene expressions were analysed by RT-PCR (Biorad-Touch real time PCR,
CA, USA) for the enzymes.