Vicks VapoRub ingredients

VVRIs (kindly provided by Procter & Gamble) were screened both individually and in combination. For individual screening of VVRIs, eucalyptus oil and turpentine oil were used at stock concentrations (4.54 M and 7.34 M, respectively). Menthol and camphor were diluted in absolute ethanol to a stock concentration of 5 M. For mixed screening, combinations of three or four VVRIs were utilized, and both combinations contained menthol, eucalyptus oil and camphor, with the addition of turpentine oil in four VVRIs. Mixed VVRIs were combined to make a stock concentration of 4.5 M. Subsequent dilutions of all VVRIs were made in calcium signalling buffer (1× HBSS, 4.2 mM sodium bicarbonate, 20 mM HEPES, 1.5 mM calcium chloride, pH 7.4) or DMEM. Equivalent vehicle concentrations of absolute ethanol in calcium signalling buffer or DMEM were utilized throughout as a control. Vehicle concentration for menthol and camphor were equivalent to 0.2% or 0.002% v/v ethanol, all other dilutions were equivalent to 0.02% or 0.0002% v/v ethanol for 10 mM and 100 μM, respectively. VVRIs were utilized throughout the study as both agonists and antagonists. Agonist usage aimed to identify whether the VVRIs cause any immediate activation of the TRP receptors. Antagonist usage aimed to identify whether pre-incubation with the VVRIs or individual ingredient could prevent subsequent activation of the TRP receptor when challenged with either a specific TRP receptor agonist or 33% hypotonic solution.