Histamine his

All reagents were applied without purification. Histamine (His) (≥99%) was purchased from Sigma Aldrich Sdn. Bhd. Cadaverine (Cad), putrescine (Put), tyramine (Tyr), heptylamine (Hep), and spermidine (Spe) were bought at 99% purity and purchased from Sigma Aldrich Sdn. Bhd. Potassium hexacyanoferrate III (K₃ [Fe(CN₆]) and potassium hexacyanoferrate II-3-hydrate (K₄ [Fe(CN₆] 3H₂O) were obtained from Merch Sdn. Bhd. Sulfuric acid (H₂SO₄), ≥97%, and lithium perchlorate were purchased from Sigma Aldrich, and polyurethane (PU) was produced by Munir et al. [36 (link)]. The phosphate buffer solution (PBS) was obtained by the combination of sodium dihydrogen phosphate (Na₂H₂PO₄) and disodium hydrogen phosphate (Na₂HPO₄), and both reagents were purchased from Merck, Sdn. Bhd. Ultrapure Milli-Q water was applied throughout.

Cells were washed twice with HBSS (Gibco) and subsequently imaged in HBSS in the dark at 37°C. Histamine (His; Sigma-Aldrich), pyrilamine (Pyr; Sigma-Aldrich), BAPTA (Cayman Chemical Company), EGTA (Sigma-Aldrich), ionomycin (Iono, LC Laboratories), Gö6983 (Calbiochem), PMA (Calbiochem), and CNO (Fisher) were added as indicated.
Epifluorescence imaging was performed on a Zeiss Axiovert 200M microscope (Carl Zeiss) equipped with a xenon lamp, a 40×/1.3 NA objective, and a cooled CCD controlled by METAFLUOR 7.7 software (Molecular Devices). For the Zeiss Axiovert 200M, the following excitation/emission filter combinations (maxima/bandwidths in nm) were used: BFP—EX380/10, EM475/25; CFP—EX420/20, EM475/25; YFP—EX495/10, EM535/25; RFP—EX568/55, EM653/95; CFP/YFP FRET—EX420/20, EM535/25. Exposure times were either 50 ms (for yellow channel), 100 ms (for red channel), or 500 ms (for all other channels), and images were acquired every 30 s. All epifluorescence experiments were subsequently analyzed using MetaFluor software. Pseudocolor images were generated in Image J.

The following reagents were used: allyl-isothiocyanate, (AITC, Sigma), 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (BCTC, Sigma), bradykinin (BK), capsaicin (Caps, Alomone Labs), ω-conotoxin GIVA (Ascent scientific), forskolin (Fors, Abcam), HC-030031 (Sigma), Histamine (His, Sigma), nifedipine (Nif, Sigma), oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC, Hycultech), PGPC (Avanti Polar Lipids), prostaglandin E2 (PGE₂, Sigma), tetrodotoxin (TTX, Ascent scientific). Dimethyl sulfoxide, chloroform or aqueous physiological solutions served as solvents.