Lacritin IP3 Assay¶
Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States
Lacritin IP3 Assay based on Webb et al. (Eur. J. Biochem. 234:714-722,1995)
- Grow cells to confluence in serum-containing medium. Wash twice with inositol-free medium.
- Incubate in serum-free/inositol-free medium containing 8 µCi/ml of 2-[3H]myo-inositol for 24 h at 37°C.
- Wash five times with HBSS supplemented with 10 mM HEPES, 3.5 mM NaHCO3, 10 mM LiCl, 1 mM MgCl2 and 1 mM CaCl2 pH 7.4.
- Incubate in the same wash solution at 37°C for 15 min.
- Replace with the same wash solution containing 10 nM LACRT or C-25 or 100 µM CCH for 0, 15, 30 and 60 sec.
- Add the samples to 2ml Dowex AG1-X8 columns. Wash off free inositol with water, then elute InsP with 0.2 M ammonium formate/0.1 M formic acid; InsP2 with 0.4 M ammonium formate/0.1 M formic acid; and InsP3 with 1.0 M ammonium formate/0.1 M formic acid.
- Measure radioactivity in scintillation counter.
Processing was as described by Webb et al. (Eur. J. Biochem. 234:714-722,1995)
Donna J. Webb, Isa M. Hussaini, Alissa M. Weaver, Tara L. Atkins, Charleen T. Chu, Salvatore V. Pizzo, Gary K. Owens, Steven L. Gonias Activated alpha2-Macroglobulin Promotes Mitogenesis in Rat Vascular Smooth Muscle Cells by a Mechanism that is Independent of Growth-Factor-Carrier Activity European Journal of Biochemistry (1995) 10.1111/j.1432-1033.1995.714_a.x