Agonist-induced production of IP₃ in PMN is measured by use of the competitive radiobinding assy. PMN (2 x 10⁶-10⁷) in 0.2 mL of phosphate-buffered saline, pH 7.4 [NaC1 (138 mM), Na₂HPO₄ (8.1 mM), KH₂PO₄ (1.5 mM), KCI (2.7 mM), CaCl₂ (1.0 mM), MgC1₂ (1.0 mM) and glucose (0.1%, w/v)] are incubated in conical polypropylene tubes at 37°C in a shaking water bath. U-73122 or U-73343 is added (in 1 μL of DMSO) 3 min before the addition of agonist, FMLP (0.1 μM) plus cytochalasin B (5 μg/mL). FMLP and cytochalasin B are added in 1 μL each of DMSO and ethanol, respectively. Appropriate vehicle controls are included in each experiment. PMN incubation mixtures are quenched with the addition of 0.07 mL of ice-cold TCA (20%, w/v) and a portion (0.2 mL) of the TCA extract is processed for the measurement of IP₃ by competitive radiobinding as described above for platelets.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Hamsters are hormone-primed with 17β-oestradiol at h?0 and progesterone at h?45. At h?48, hamsters are pretested for motor behaviour, followed by sexual behaviour testing, and bilateral infusions of U73122 (400 nM/μL) or saline vehicle. Thirty minutes after infusions, hamsters are re-tested for sexual behaviour (post inhibitor infusion test) and, immediately after testing, infused bilaterally with SKF38393 (100 ng/μL), muscimol (100 ng/μL), or saline vehicle. Thirty minutes after the agonist or vehicle infusions, lordosis and motor behaviour of hamsters is reassessed (post agonist infusion test). All hamsters are assigned to one pretreatment condition, U73122 or vehicle, and are tested once a week for 3 weeks until all infusion conditions (SKF38393, muscimol or vehicle), are received. The order in which hamsters receive SKF38393, muscimol or vehicle infusions is counterbalanced across the group.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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