Bodipy pc

Two microliters of the solution of fluorescently-labeled substrate Bodipy-phosphatidylcholine (2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) amino) undecyl) sn-glycero-3-phosphocholine) (Bodipy-PC, D-3771, Invitrogen) in ethanol was added to the pollen culture (2 mg/mL) at indicated times (final concentration of Bodipy-PC was 0.66 µg/mL). After incubation on an orbital shaker at RT for 10 min, lipids were extracted as described above, the organic phase was evaporated to dryness by a vacuum evaporator, redissolved in ethanol and analyzed using thin-layer chromatography as described earlier (Pejchar et al., 2010 (link)). Briefly, samples were applied on the HP-TLC silica plates by automatic sampler, plates were developed in a mobile phase chloroform: methanol: water 65: 25: 4 (v/v/v), dried and scanned by FLA-7000 (Fujifilm) laser scanner. Individual spots were identified based on the comparison with fluorescently-labeled lipid standards and quantified by Multi Gauge (Fujifilm) software.

We visualized mucin by labelling it with an Atto590 N-hydrosuccinimide ester dye (λ_ex\λ_em = 594/624 nm, Sigma-Aldrich^®). Briefly, we added 100 mg of mucin in 20 ml phosphate buffer (100 mM pH 8.5) to 1 mg of the dye dissolved in amine-free dimethylformamide (Molecular Biology Grade, Sigma-Aldrich^®). We continually stirred the solution for 2 h at 22°C. Then, we added nine parts by volume of previously cooled aqueous ethanol solution (90% v/v) at −80°C to the suspension to induce precipitation of the mucin. We precipitated mucin overnight by maintaining the temperature of the suspension at −20°C. We recovered the precipitate by centrifuging the suspension at 50 000g for 15 min. We then washed the precipitate with cold ethanol (−80°C) to remove as much of the residual Atto590 dye, which is highly soluble in ethanol, as possible. We resuspended the dried mucin in 20 ml nanopure water and dialysed it overnight to remove phosphate ions that originated from the buffer. To visualize DPPC, we added 100 µg boron-dipyrromethene-phosphocholine (BODIPY-PC, λ_ex\λ_em = 488\503 nm, Molecular Probes, Life Technologies Inc.) or 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC, λ_ex\λ_em = 460\534 nm, Avanti Polar Lipids Inc.) to unlabelled DPPC.