Nbd phallacidin

F-actin was measured in unfractionated leukocytes prepared as described above. The leukocytes were first incubated for 30 min on ice with allophycocyanin-labeled anti-CD49d (BioLegend, San Diego, CA). After washing with PBS, the cells were suspended in PBS containing Ca⁺⁺ (1.8 mM) and Mg⁺⁺ (1 mM) at a concentration of 5.5 × 10⁶ cells/ml. Aliquots (100 μl) of the fluorescently labelled cells were then preincubated for 5 min at 37 °C with vehicle (1 μl DMSO) or different concentrations of 53, 34, or Gue1654, followed by addition of either vehicle (10 μl PBS containing Ca⁺⁺, Mg⁺⁺, and 0.1% BSA) or 5-oxo-ETE (final concentration, 10 nM). After 20 s, the incubations were terminated by addition of formaldehyde (37%) to give a final concentration of 8.5%, and kept on ice for 30 min. A mixture of lysophosphatidylcholine (30 μg in 23.8 μl PBS) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD-phallacidin; Molecular Probes; 49 pmol in 6.2 μl methanol; final concentration, 0.3 μM) was added to each sample ^{33 (link)}, followed by incubation overnight in the dark at 4 °C. Immediately prior to data acquisition by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA), 200μl PBS containing 1% formaldehyde was added to each sample. Eosinophils were identified by high side scatter and high expression of CD49d, whereas neutrophils displayed high side scatter but low CD49d expression.