Fluo 8 no wash calcium assay kit

Fully differentiated human moDCs were seeded into 96-well plates at 5 × 10⁵ cell per mL in serum-free AIMV medium. Alterations in intracellular Ca²⁺ concentration by the 5-HT_2B agonist BW723C86 were assessed by the Fluo-8 No Wash Calcium Assay kit (Abcam) according to the manufacturer’s instructions. Fluorescence intensities were measured in a TriStar² LB 942 Multidetection Microplate Reader (Berthold Technologies, Bad Wildbad Germany). Samples were excited at 485 nm and Ca²⁺-bound Fluo-8 photon emission was recorded at 525 nm. Relative Fluo-8 fluorescence was expressed as F_max/F₀ (F_max = maximum fluorescence; F₀ = baseline fluorescence).

Intracellular free Ca²⁺ was measured using the Fluo-8-no wash calcium assay kit (Abcam). Fluo-8 epifluorescence was excited at 490 nm and images were obtained at 520 nm. The imaging data were collected with a fluorescence microscopy system (Olympus BX51), and the intensity of fluorescence was determined with a fluorometer (Bio-Tek Instruments).

Intracellular calcium mobilization was monitored using the Fluo-8 No Wash Calcium Assay Kit (Abcam) according to the manufacturer’s instructions for non-adherent cells. Healthy donor neutrophils isolated as described above were resuspended in equal amounts of HBSS supplemented with 1.26 mM CaCl₂ and 20 mM HEPES and Fluo-8 dye-loading solution. A total of 10⁵ cells/well/100 μl were seeded into sterile, black, flat-bottom 96-well plates and incubated for 30 min. Pre-warmed HBSS supplemented with 1.26 mM CaCl₂ was added to a final assay volume of 270 μl. The plate was transferred into a fluorescence plate reader with a dual injector function (Varioskan Flash, Thermo Scientific) and monitored at 37°C for 85 min reading fluorescence at an excitation wavelength of 490 nm and an emission wavelength of 525 nm. After baseline recording, MCP-1 was injected at medium speed (BioLegend, 0.5 ng/ml final concentration) and fluorescence was measured per well immediately afterward. The plate was scanned every minute for 20 min. Then, ionomycin was injected at medium speed (Sigma, 1.3 μM final concentration) and fluorescence was again measured per well immediately afterward. Calcium mobilization was monitored for 15 min every minute and then for 50 min every 5 min. Results are presented as fold change to baseline.

1,2-bis-(o-Aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) was purchased from Calbiochem (Darmstadt, Germany). Trypsin (without EDTA) was purchased from Invitrogen (Darmstadt, Germany). U73122, U73343, 2-APB, BAPTA, CaCl₂ and EGTA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fluo-8 No Wash Calcium Assay Kit was purchased from Abcam (Cambridge, UK). All other chemicals were obtained from Roth (Karlsruhe, Germany).