Fluo 4

Manufactured by Dojindo Laboratories

Sourced in Japan

Fluo-4 is a fluorescent calcium indicator dye produced by Dojindo Laboratories. It is a small-molecule chemical compound that exhibits a significant increase in fluorescence intensity upon binding to calcium ions. Fluo-4 can be used to measure changes in intracellular calcium levels in various cell types and biological systems.

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13 protocols using fluo 4

Nicotine-Induced Calcium Flux in H1299 Cells

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Cells (1×10⁵ cells/ml), including untransfected and shRNA-transfected H1299 cells, were seeded onto glass chamber slides (043320B; Shengyou Biotechnology, Hangzhou, China). Cells were cultured for 24 h, and the medium was then removed and replaced with Hanks balanced salt solution (HBSS) containing 1 µM Fluo-4 (cat no. F312; Dojindo Laboratories Inc.), after which the cells were incubated for 1 h at 37°C. Cells were then washed once with HBSS and stored in fresh HBSS for 30 min prior to further experimentation. Nicotine was directly added into the chamber at a final concentration of 1 mM. In some chambers, the cells were pre-incubated with α-BTX at a final concentration of 1 µM for 30 min at 37°C prior to the addition of nicotine. Immediately after the addition of nicotine, Fluo-4 was excited with an Argon laser (the excitation wavelength was 494 nm and emission wavelength was 519 nm) using a Zeiss LSM-710 EXCITER microscope (Zeiss, Thornwood, NY, USA) to assess changes in the calcium flux from the nAChR ion channels. The fluorescence intensity of calcium was recorded as the ratio of F and F0, where F represented the peak fluorescence intensity of the cellular calcium influx when stimulated by nicotine, and F0 represented the basic fluorescence intensity of cellular calcium influx. The mean relative fluorescence of the peak was calculated as [(F-F0)/F0] × 100%

Zhang C., Yu P., Zhu L., Zhao Q., Lu X, & Bo S. (2017). Blockade of α7 nicotinic acetylcholine receptors inhibit nicotine-induced tumor growth and vimentin expression in non-small cell lung cancer through MEK/ERK signaling way. Oncology Reports, 38(6), 3309-3318.

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Measurement of Intracellular Calcium Levels

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Intracellular calcium levels were measured using a Calcium kit Fluo 4 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions^{45 (link),46 (link)}. BMMCs were seeded at 4.0 × 10⁴ cells/well in a 96-well black culture plate and sensitised with anti-DNP IgE (50 ng/mL) at 37 °C overnight. After washing with PBS, the cells were incubated with Fluo 4-AM for 1 h at 37 °C. Cells were again washed with PBS, treated with the recording buffer containing Eucalyptus oil and 1,8-cineole, and then incubated at 37 °C for 30 min. Next, the cells were stimulated with DNP-HSA (50 ng/mL), and the fluorescence intensity was immediately monitored with an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a microplate reader (BioTeh, Winooski, St, USA).

Nakamura T., Yoshida N., Yamanoi Y., Honryo A., Tomita H., Kuwabara H, & Kojima Y. (2020). Eucalyptus oil reduces allergic reactions and suppresses mast cell degranulation by downregulating IgE-FcεRI signalling. Scientific Reports, 10, 20940.

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Measuring FFAR1 receptor activation in CHO cells

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Chinese hamster ovary (CHO) cells stably expressing human FFAR1 with different receptor mRNA expression levels (clones #104 and #2) [32 (link)] were cultured with minimum essential medium-alpha (FUJIFILM Wako) containing 10% dialyzed fetal bovine serum (FBS, GE Healthcare Life Sciences, Buckinghamshire, UK), 10 mM HEPES (Thermo Fisher Scientific, Waltham, MA), 100 IU/mL penicillin, and 100 μg/mL streptomycin in 5% CO₂ at 37°C. Cells (1 x 10⁴ cells /well) were seeded in 384 well plates and then incubated overnight. After removing of the medium, cells were incubated in 30 μL of loading buffer (Hanks' Balanced Salt Solution containing 20 mM HEPES, 0.1% fatty acid-free BSA (FUJIFILM Wako), 0.08% Pluronic F127 (#CSK-01F, Dojindo Kumamoto, Japan), 2.5 mmol/L Probenecid (#CSK-03F, Dojindo) and 2.5 μg/mL Fluo4 (#F311, Dojindo)) for 60 min in 5% CO₂ at 37°C. Test compounds at various concentrations were added into the cells and increase of the intracellular Ca²⁺ concentration was monitored by the FLIPR Tetra system (Molecular Devices, Tokyo, Japan) for 180 sec. EC₅₀ was calculated by data analysis using a 4-parameter logistic equation in Graphpad Prism 7 software.

Ueno H., Ito R., Abe S.I., Ogino H., Maruyama M., Miyashita H., Miyamoto Y., Moritoh Y., Tsujihata Y., Takeuchi K, & Nishigaki N. (2019). GPR40 full agonism exerts feeding suppression and weight loss through afferent vagal nerve. PLoS ONE, 14(9), e0222653.

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Astragaloside IV Effects on Cellular Calcium

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Astragaloside IV (purity at 98%) was purchased from Shanghai Bogoo Biotechnology Company (Shanghai Co. Ltd., China). Palmitic acid, BAPTA-AM and SKF96365 were obtained from Sigma–Aldrich (St. Louis, MO, USA). Mag-Fura-2, Rhod-2 and JC-1 were purchased from Invitrogen (Carlsbad, CA, USA). Fluo-4 was purchased from Dojindo (Kumamoto, Japan) and OAG was purchased from MedChem Express (Monmouth Junction, NJ, USA).

Zang Y., Liu S., Cao A., Shan X., Deng W., Li Z., Wang H., Wang Y., Wang L, & Peng W. (2021). Astragaloside IV inhibits palmitic acid-induced apoptosis through regulation of calcium homeostasis in mice podocytes. Molecular Biology Reports, 48(2), 1453-1464.

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Intracellular Calcium Imaging in INS-1 Cells

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Intracellular calcium concentration ([Ca²⁺]i) in INS‐1 cells was measured with a Calcium kit Fluo‐4 (#CS22; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). INS‐1 cells were placed on a φ35‐mm glass bottom culture dish. Growth medium was replaced with loading buffer containing 3 mmol/L glucose and Fluo4‐AM, and the culture dish was incubated at 37°C for 1 h. Loading buffer was then replaced with recording buffer containing 3 mmol/L glucose. The Fluo‐4 fluorescent signal over the INS‐1 cell area was detected through excitation at 488 nm using an LSM 880 Live microscope (Carl Zeiss, Oberkochen, Germany).

Udagawa H., Hiramoto M., Kawaguchi M., Uebanso T., Ohara‐Imaizumi M., Nammo T., Nishimura W, & Yasuda K. (2020). Characterization of the taste receptor‐related G‐protein, α‐gustducin, in pancreatic β‐cells. Journal of Diabetes Investigation, 11(4), 814-822.

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Characterization of A20 B cell lymphoma

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A20 cell line, derived from mouse B cell lymphoma on BALB/c background [13 (link)], was obtained from American Type Culture Collection (Manassas, VA). Primary cells were isolated from bone marrow (BM), spleen (SPL) or peripheral blood (PB) of B6 by standard methods. The A20 transfectants or primary cells were stained with the following antibodies (Abs): anti-mouse CEACAM1 mAb, CC1 (kindly provided by Dr. Kathlyn Holmes, University of Colorado, CO), anti-IgG1, FITC-anti-B220, PE-anti-IgM, FITC-anti-CD4, PE-anti-CD3ε, PerCP-anti-B220, PE-anti-CD43, PE-anti-CD25, PE-anti-Igκ, PE-anti-CD138, PE-anti-CD5, PE-anti-H-2K^d, PE-anti-I-A^d, PE-anti-CD69, PE-anti-CD80 and PE-anti-CD86 Abs (BD Biosciences, San Jose, CA). Data acquisition was performed using FACS Calibur^™ and analysis software Cell Quest^™ (BD Biosciences).
In some experiments, the A20 transfectants were treated with Fluo-4® (Dojindo, Kumamoto, Japan), and applied for FACS to analyze intracellular Ca²⁺ influx. During the acquisition, cells were treated with anti-Igκ (Southern Biotech, Birmingham, AL) to stimulate BCR signaling, and kinetics for Ca²⁺ influx were measured at FL2.

Tsugawa N., Yamada D., Watabe T., Onizawa M., Wang S., Nemoto Y., Oshima S., Tsubata T., Adachi T., Kawano Y., Watanabe M., Blumberg R.S., Okamoto R, & Nagaishi T. (2020). CEACAM1 specifically suppresses B cell receptor signaling-mediated activation. Biochemical and biophysical research communications, 535, 99-105.

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Quantifying Receptor-Mediated Calcium Signaling

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Chinese hamster ovary (CHO)‐K1 cells expressing M₁R–M₅R were plated on a 96‐well black, clear bottom plate at 30 000 cells/well and incubated at 37°C in an atmosphere of 5% CO₂ for 1 day. On the day of the assay, cells were incubated with calcium dye buffer (Hanks’ Balanced Salt Solution (HBSS) containing 20 mmol/L HEPES, 0.1% fatty acid‐free bovine serum albumin (BSA), 0.08% pluronic F127 (Dojindo Laboratories), 2.5 μg/mL Fluo‐4 (Dojindo Laboratories), and 1.25 mmol/L probenecid (Dojindo Laboratories)) for 30 minutes at 37°C in an atmosphere of 5% CO₂ and then incubated for 30 minutes at room temperature. To measure Ca²⁺ mobilization using CellLux (PerkinElmer), cells were stimulated with T‐495 or MK‐7622 (0.01‐1000 nmol/L for PAM activity; 0.3‐10 000 nmol/L for agonist activity) in assay buffer (HBSS containing 20 mmol/L HEPES and 0.1% fatty acid‐free BSA) with or without an EC₂₀ concentration of ACh. The inflection point (IP) and EC₅₀ values were calculated using the following equation by GraphPad Prism 5 software (GraphPad Software Inc):

Y = Bottom + \frac{(Top - Bottom)}{1 + 10^{((Log IP orEC 50 - X) \times HillSlope))}}

where X and Y are the log concentration of a compound and the percentage of Ca²⁺ response, respectively, and Top and Bottom are the upper and lower plateaus, respectively.

Mandai T., Sako Y., Kurimoto E., Shimizu Y., Nakamura M., Fushimi M., Maeda R., Miyamoto M, & Kimura H. (2020). T‐495, a novel low cooperative M1 receptor positive allosteric modulator, improves memory deficits associated with cholinergic dysfunction and is characterized by low gastrointestinal side effect risk. Pharmacology Research & Perspectives, 8(1), e00560.

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Measuring Cytosolic Calcium Dynamics

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To estimate changes in cytosolic Ca²⁺, SK-N-SH cells were loaded with the Ca²⁺-sensitive fluorescent dye Fluo-4 (Dojindo Laboratories, Kumamoto, Japan) and florescence intensity (correlated with Ca²⁺) was analyzed by flow cytometry or confocal microscopy. Cells were transferred to microcentrifuge tubes 24 h after transfection with or without BAPTA/AM (50 μM for 1 h), then washed three times in Hank’s Balanced Salt Solution (HBSS) and incubated in Fluo-4/AM (1 μM) for 30 min in a 37°C incubator with 5% CO₂. Cells were then centrifuged at 3000 rpm for 5 min and washed three times in HBSS. The green fluorescence intensity (516 nm) was quantified using a FACSAria flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). For confocal imaging, cells were seeded on poly-l-lysine-coated glass cover slips 24 h after transfection with or without BAPTA/AM (50 μM for 1 h), as described above. Images were acquired with a confocal microscope.

Yang W., Wang X., Liu J., Duan C., Gao G., Lu L., Yu S, & Yang H. (2017). PINK1 suppresses alpha-synuclein-induced neuronal injury: a novel mechanism in protein phosphatase 2A activation. Oncotarget, 9(1), 37-53.

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Quantifying DRG Neuron Calcium Response to Capsaicin

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DRG neurons were seeded in 96-well cell culture plates at a density of 1.5 × 10⁴ cells per well and cultured overnight. Intracellular calcium ion responses to capsaicin were measured using the calcium kit II Fluo-4 (CS32, Dojindo) in accordance with the manufacturer’s instructions. The temperature of the platform was set to 37 °C. Cells were fluorescently imaged at an excitation wavelength of 495 nm every 7 s, and the fluorescence intensities of neurons were quantified at 515 nm. Fluorescence intensities of neurons were quantified simultaneously for the entire well. Capsaicin (1 μM) was added to measure the response.

Tanaka T., Okuda H., Isonishi A., Terada Y., Kitabatake M., Shinjo T., Nishimura K., Takemura S., Furue H., Ito T., Tatsumi K, & Wanaka A. (2023). Dermal macrophages set pain sensitivity by modulating the amount of tissue NGF through an SNX25–Nrf2 pathway. Nature Immunology, 24(3), 439-451.

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Intracellular Calcium Dynamics in Mast Cells

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Intracellular calcium levels were measured using a Calcium kit Fluo 4 (Dojindo Laboratories, Mashiki, Japan) according to the manufacturer’s instructions. RBL-2H3 cells were seeded at 4.0 × 10⁴ cells/well in a 96-well black culture plate (Sumitomo Bakelite, Tokyo, Japan) and sensitized with anti-DNP IgE (50 ng/mL) at 37 °C overnight. After washing with PBS, the cells were incubated in 100 μL of Fluo 4-AM for 1 h at 37 °C. After washing with PBS, the cells were treated with 100 μL of the recording buffer containing 34, 67, or 135 µM DHEA or 0.1% (v/v) ethanol (vehicle) and incubated at 37 °C for 30 min. The cells were then stimulated with DNP-HSA (50 ng/mL), and the fluorescent intensity was immediately monitored with an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a microplate reader (Infinite 200 PRO, Tecan, Männedorf, Switzerland).

Nishi K., Kanayama Y., Kim I.H., Nakata A., Nishiwaki H, & Sugahara T. (2019). Docosahexaenoyl ethanolamide mitigates IgE-mediated allergic reactions by inhibiting mast cell degranulation and regulating allergy-related immune cells. Scientific Reports, 9, 16213.

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