Indo 1 acetoxymethyl ester

Activation of CD8+ T cells following ligation of BLT1 by LTB4 was monitored by changes in intracellular Ca²⁺ concentrations. Isolated CD8+ T cells (5 ×10⁶ cells/mL) were incubated for 45 min at 37°C with 5 μM indo-1 acetoxymethyl ester (Invitrogen, Carlsbad, CA). Cells were resuspended and subjected to analysis by flow cytometry (LSR II; Becton Dickinson, Franklin Lakes, NJ) with FlowJo software (16 (link)). After a baseline was established, cells were stimulated with LTB4 (100 nM, 1000 nM, Sigma-Aldrich) and intracellular Ca²⁺ concentrations were determined by measurement of the median fluorescence ratio of indo-1 acetoxymethyl ester at 390 nm/490 nm emission.

Neutrophils were loaded with indo-1 acetoxymethyl ester (Invitrogen, Carlsbad, CA) as described previously.^{10 (link)} The cells were then washed and resuspended in PBS. Five minutes before commencing data acquisition, the cells were placed in a cuvette and Ca⁺⁺ and Mg⁺⁺ were added to give final concentrations of 1.8 and 1 mM, respectively. The cuvette was then placed in a Deltascan 4000 spectrofluorometer (Photon Technology International, Birmingham, NJ) in a cuvette holder maintained at 37 °C and equipped with a magnetic stirrer. Fluorescence was monitored at excitation and emission wavelengths of 331 and 410 nm, respectively, and the baseline allowed to stabilize before addition of either vehicle (10 μl DMSO) or potential OXE receptor antagonist. Two min later, 5-oxo-ETE (final concentration 10 nM in PBS containing 0.1% BSA) was added, followed 1 min later by addition of digitonin (final concentration, 0.1%) to permit measurement of maximal fluorescence. In some cases a second agonist (LTB₄, PAF, fMLP, or IL-8) was added 2 min after addition of 5-oxo-ETE, followed 1 min later by digitonin. Calcium levels were calculated as previously described.^{10 (link)}

TCL1-192 cells (5×10⁶ cells/mL) were incubated in dye-free RPMI1640 medium supplemented with 5% FBS and 1 μM fluorescent Ca⁺⁺ indicator Indo-1, acetoxymethyl ester (Invitrogen). 1×10⁶ cells per tube were run for 30 seconds on LSRII to obtain a baseline reading, and then stimulated for 5 minutes by 5 μg/ml F(ab′)₂ fragments of anti-IgM polyclonal antibodies or 200 ng/ml CXCL12. Kinetic data were analyzed with FlowJo (BD Biosciences). The intracellular Ca⁺⁺ levels were calculated from the violet (440 nm) and green (530nm) emissions and plotted against the Time parameter.

BMDCs (4×10⁶ cells/mL) were loaded for 45 minutes at 37°C with 5 μM indo-1 acetoxymethyl ester (Invitrogen, Carlsbad, CA) and preincubated with loratadine and/or JNJ7777120 in RPMI 1640 medium containing 2% FCS. Histamine-induced intracellular Ca2⁺ mobilization was determined as previously described (26 (link)).