Fluo‑3 Calcium Ion Detection Kit is a kit for detecting intracellular calcium ion concentration based on the Fluo‑3 AM calcium fluorescent probe. This kit can be used for fluorescence imaging via fluorescence microscopy, as well as for quantitative fluorescence detection using a fluorescence microplate reader or flow cytometry. Dynamic changes in intracellular calcium ion concentration can also be detected using fluorescence microscopy or a fluorescence microplate reader.Calcium is generally the most abundant mineral in mammalian organisms, involved in the regulation of numerous cellular processes and is one of the most important intracellular regulatory factors. Calcium exists either as free ions or in calcium‑bound complexes; for instance, calcium phosphate and calcium carbonate complexes are major components of bone tissue. A wide range of physiological processes are regulated by calcium signaling, including muscle contraction, cell adhesion, hormone/neurotransmitter release, glycogen metabolism, cell proliferation/differentiation, blood coagulation, neuronal or synaptic transmission, and structural support of bone. Defects in the integrity of cell‑specific calcium signaling systems can lead to the development of various diseases[1].The principle of this kit for detecting intracellular calcium flux is as follows: the calcium fluorescent probe Fluo‑3 AM itself exhibits almost no fluorescence. Once inside cells, it is hydrolyzed by intracellular esterases to form Fluo‑3, which emits green fluorescence upon binding calcium ions. Influx of extracellular calcium or release of calcium from intracellular stores increases cytosolic calcium concentration, allowing more calcium binding to intracellular Fluo‑3 and ultimately enhancing intracellular green fluorescence intensity. Efflux or sequestration of intracellular calcium reduces cytosolic calcium concentration, decreasing calcium available for Fluo‑3 binding and thereby lowering green fluorescence intensity. The calcium fluorescent probe Fluo‑3 AM is widely used for detecting dynamic changes in calcium concentration and for high‑throughput screening of G‑protein‑coupled receptor agonists or antagonists that affect calcium mobilization.This kit is convenient to use and provides a complete set of supporting reagents, with no washing required after staining. The kit includes Assay Buffer for diluting Fluo‑3 AM, which greatly preserves cell viability and near‑normal growth conditions. It also provides Solubility Enhancer to improve the solubility of Fluo‑3 AM and enhance staining performance. The included Staining Enhancer effectively retains intracellular fluorescence and reduces background fluorescence during detection. Meanwhile, no washing step is needed after staining, making the kit very convenient to use.For samples in 96‑well plates, the kit package supports 200 tests based on 100 μL Fluo‑3 Staining Solution per well. For flow cytometry assays with a 0.5 mL detection system per sample, the kit supports 40 tests. For adherent cultured cells in 6‑well plates, the kit supports 20 tests based on 1 mL Fluo‑3 Staining Solution per well.Precautions1. Fluo‑3 AM is highly unstable in water; appropriate aliquots are recommended upon first use.2. Fluo‑3 AM may solidify at low temperatures such as 4 ℃ or on ice and adhere to the bottom, wall, or cap of the centrifuge tube. Warm briefly in a 20–25 ℃ water bath until fully dissolved, centrifuge briefly to collect at the bottom, then use.3. The working concentration of Fluo‑3 AM, cell amount, incubation temperature, and time can be optimized according to the cell type and specific stimuli applied. For example, the probe concentration can be adjusted appropriately between 0.2× and 2×.4. Ensure Fluo‑3 AM (500×) and Solubility Enhancer (500×) are completely dissolved before use.5. All fluorescent dyes are subject to photobleaching; protect from light as much as possible to slow fluorescence decay.6. All products of this company are for research use only by professional personnel in life science research. They are not intended for clinical diagnosis or therapy, food or drug applications, or storage in ordinary residential premises.7. All products must be operated by qualified professional technicians wearing masks, gloves, and lab coats, in compliance with biological laboratory safety protocols.Protocol1. Preparation of Fluo‑3 Staining SolutionPrepare an appropriate volume of Fluo‑3 Staining Solution according to the system of 100 μL per well in 96‑well plates, referring to the table below, and mix thoroughly.Reagent1 Sample10 Samples100 SamplesBioFluo-3 AM0.2 μL2 μL20 μLSolubility Enhancer (500×)0.2 μL2 μL20 μLAssay Buffer99.6 μL996 μL9.96 mLBioFluo-3 Staining Solution100 μL1 mL10 mLNote 1: Prepared Fluo‑3 Staining Solution must be used immediately in one run and cannot be frozen. The concentration of Fluo‑3 AM in the staining solution can be adjusted between 0.2× and 2× based on staining performance.Note 2: Other suitable solutions such as serum‑free medium, HBSS, or PBS may also be used to dilute Fluo‑3 AM (500×). The Assay Buffer provided with this kit effectively maintains normal cell status and provides partial nutrition over time, generally yielding better results than PBS or HBSS.Note 3: Staining Enhancer may have certain effects on cell function and is unnecessary for most cells. It is recommended for cells (e.g., CHO) showing obvious extrusion of de‑esterified fluorescent Fluo‑3. Add Staining Enhancer (100×) to Fluo‑3 Staining Solution at a dilution of 1:100 to prepare solution containing 1× Staining Enhancer. Cell incubation time in solution containing 1× Staining Enhancer should not exceed 2 hours.2. Detection by Fluorescence Microscopy(1) Seeding and culture. Seed cells in multi‑well plates (e.g., 96‑well plates), cell culture dishes, or cell coverslips. Treat cells as required by experimental design.(2) Washing (optional). For adherent cells, remove culture medium and wash cells once with PBS. For suspension cells, centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant, and wash once with PBS. Phenol red or serum may interfere with detection; a vacuum pump is recommended for removing medium and PBS. Washing may be omitted if residual liquid can be thoroughly removed. One wash generally reduces background fluorescence, but good staining can be achieved without washing.(3) Staining. Add an appropriate volume of Fluo‑3 Staining Solution: typically 100 μL per well for 96‑well plates, 250 μL for 24‑well plates, 500 μL for 12‑well plates, and 1 mL for 6‑well plates. Incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted between 10 and 60 min.Note 1: For initial experiments with uncertain temperature and time, test incubation at 37 ℃ for 30 min first. Shorten time or lower temperature if excessive cell death occurs; extend time if fluorescence intensity is too weak.Note 2: Cells may be washed 1–3 times with PBS or HBSS after incubation.(4) Detection. After incubation, observe staining under a fluorescence microscope (Fluo‑3 AM emits green fluorescence, Ex/Em = 490/525 nm).3. Detection by Flow Cytometry(1) Cell preparation. Harvest adherent cells by trypsin digestion, resuspend in medium, and wash once with PBS. For suspension cells, centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant, and wash once with PBS. Recommended cell amount per sample is 10⁶ cells.(2) Staining. To the pellet of 10⁶ cells from the previous step, add 1 mL Fluo‑3 Staining Solution and resuspend into a single‑cell suspension. Incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted between 10 and 60 min.Note 1: For initial experiments with uncertain temperature and time, test incubation at 37 ℃ for 30 min first. Shorten time or lower temperature if excessive cell death occurs; extend time if fluorescence intensity is too weak.Note 2: Cells may be washed 1–3 times with PBS or HBSS after incubation.(3) Detection. After incubation, analyze directly by flow cytometry, or centrifuge at 250–1000×g for 5 min at room temperature to pellet cells, remove liquid, resuspend in 0.5 mL Assay Buffer per sample, and analyze (Fluo‑3 AM emits green fluorescence, Ex/Em = 490/525 nm).Note 1: Use unstained cells in Assay Buffer as the negative control for flow cytometry setup.Note 2: As flow cytometry is highly sensitive, the probe concentration used may be lower than for microscopy; adjust the dilution of Fluo‑3 AM according to cell type and actual staining.4. Detection by Fluorescence Microplate Reader(1) Seeding and culture. Seed cells in black 96‑well plates. Cell number per well should be controlled within 100–10,000, typically 2000–5000. Treat cells as required by experimental design.(2) Washing (optional). For adherent cells, remove culture medium and wash once with PBS. For suspension cells, centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant, and wash once with PBS. Phenol red or serum may interfere with detection; a vacuum pump is recommended for removing medium and PBS. Washing may be omitted if residual liquid can be thoroughly removed. One wash generally reduces background fluorescence, but good staining can be achieved without washing.(3) Staining. Add an appropriate volume of Fluo‑3 Staining Solution, typically 100 μL per well in 96‑well plates. Incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted between 10 and 60 min.Note 1: For initial experiments with uncertain temperature and time, test incubation at 37 ℃ for 30 min first. Shorten time or lower temperature if excessive cell death occurs; extend time if fluorescence intensity is too weak.Note 2: Cells may be washed 1–3 times with PBS or HBSS after incubation.(4) Detection. After incubation, measure using a fluorescence microplate reader (Fluo‑3 AM emits green fluorescence, Ex/Em = 490/525 nm).The effect of drug stimulation can be determined by comparing RFU (Relative Fluorescence Units) between control and treated groups.References1. Clapham DE. Cell. 2007. 131(6):1047–58.2. Wu N, Nishioka WK, Derecki NC, Maher MP. Sci Rep. 2019. 9(1):12692.3. Hagen BM, Boyman L, Kao JP, Lederer WJ. Cell Calcium. 2012. 52(2):170–81.

Specifications & Purity: Fluo‑3 Calcium Ion Detection Kit is a kit for detecting intracellular calcium ion concentration based on the Fluo‑3 AM calcium fluorescent probe. This kit can be used for fluorescence imaging via fluorescence microscopy, as well as for quantitative fluorescence detection using a fluorescence microplate reader or flow cytometry. Dynamic changes in intracellular calcium ion concentration can also be detected using fluorescence microscopy or a fluorescence microplate reader.Calcium is generally the most abundant mineral in mammalian organisms, involved in the regulation of numerous cellular processes and is one of the most important intracellular regulatory factors. Calcium exists either as free ions or in calcium‑bound complexes; for instance, calcium phosphate and calcium carbonate complexes are major components of bone tissue. A wide range of physiological processes are regulated by calcium signaling, including muscle contraction, cell adhesion, hormone/neurotransmitter release, glycogen metabolism, cell proliferation/differentiation, blood coagulation, neuronal or synaptic transmission, and structural support of bone. Defects in the integrity of cell‑specific calcium signaling systems can lead to the development of various diseases[1].The principle of this kit for detecting intracellular calcium flux is as follows: the calcium fluorescent probe Fluo‑3 AM itself exhibits almost no fluorescence. Once inside cells, it is hydrolyzed by intracellular esterases to form Fluo‑3, which emits green fluorescence upon binding calcium ions. Influx of extracellular calcium or release of calcium from intracellular stores increases cytosolic calcium concentration, allowing more calcium binding to intracellular Fluo‑3 and ultimately enhancing intracellular green fluorescence intensity. Efflux or sequestration of intracellular calcium reduces cytosolic calcium concentration, decreasing calcium available for Fluo‑3 binding and thereby lowering green fluorescence intensity. The calcium fluorescent probe Fluo‑3 AM is widely used for detecting dynamic changes in calcium concentration and for high‑throughput screening of G‑protein‑coupled receptor agonists or antagonists that affect calcium mobilization.This kit is convenient to use and provides a complete set of supporting reagents, with no washing required after staining. The kit includes Assay Buffer for diluting Fluo‑3 AM, which greatly preserves cell viability and near‑normal growth conditions. It also provides Solubility Enhancer to improve the solubility of Fluo‑3 AM and enhance staining performance. The included Staining Enhancer effectively retains intracellular fluorescence and reduces background fluorescence during detection. Meanwhile, no washing step is needed after staining, making the kit very convenient to use.For samples in 96‑well plates, the kit package supports 200 tests based on 100 μL Fluo‑3 Staining Solution per well. For flow cytometry assays with a 0.5 mL detection system per sample, the kit supports 40 tests. For adherent cultured cells in 6‑well plates, the kit supports 20 tests based on 1 mL Fluo‑3 Staining Solution per well.Precautions1. Fluo‑3 AM is highly unstable in water; appropriate aliquots are recommended upon first use.2. Fluo‑3 AM may solidify at low temperatures such as 4 ℃ or on ice and adhere to the bottom, wall, or cap of the centrifuge tube. Warm briefly in a 20–25 ℃ water bath until fully dissolved, centrifuge briefly to collect at the bottom, then use.3. The working concentration of Fluo‑3 AM, cell amount, incubation temperature, and time can be optimized according to the cell type and specific stimuli applied. For example, the probe concentration can be adjusted appropriately between 0.2× and 2×.4. Ensure Fluo‑3 AM (500×) and Solubility Enhancer (500×) are completely dissolved before use.5. All fluorescent dyes are subject to photobleaching; protect from light as much as possible to slow fluorescence decay.6. All products of this company are for research use only by professional personnel in life science research. They are not intended for clinical diagnosis or therapy, food or drug applications, or storage in ordinary residential premises.7. All products must be operated by qualified professional technicians wearing masks, gloves, and lab coats, in compliance with biological laboratory safety protocols.Protocol1. Preparation of Fluo‑3 Staining SolutionPrepare an appropriate volume of Fluo‑3 Staining Solution according to the system of 100 μL per well in 96‑well plates, referring to the table below, and mix thoroughly.Reagent1 Sample10 Samples100 SamplesBioFluo-3 AM0.2 μL2 μL20 μLSolubility Enhancer (500×)0.2 μL2 μL20 μLAssay Buffer99.6 μL996 μL9.96 mLBioFluo-3 Staining Solution100 μL1 mL10 mLNote 1: Prepared Fluo‑3 Staining Solution must be used immediately in one run and cannot be frozen. The concentration of Fluo‑3 AM in the staining solution can be adjusted between 0.2× and 2× based on staining performance.Note 2: Other suitable solutions such as serum‑free medium, HBSS, or PBS may also be used to dilute Fluo‑3 AM (500×). The Assay Buffer provided with this kit effectively maintains normal cell status and provides partial nutrition over time, generally yielding better results than PBS or HBSS.Note 3: Staining Enhancer may have certain effects on cell function and is unnecessary for most cells. It is recommended for cells (e.g., CHO) showing obvious extrusion of de‑esterified fluorescent Fluo‑3. Add Staining Enhancer (100×) to Fluo‑3 Staining Solution at a dilution of 1:100 to prepare solution containing 1× Staining Enhancer. Cell incubation time in solution containing 1× Staining Enhancer should not exceed 2 hours.2. Detection by Fluorescence Microscopy(1) Seeding and culture. Seed cells in multi‑well plates (e.g., 96‑well plates), cell culture dishes, or cell coverslips. Treat cells as required by experimental design.(2) Washing (optional). For adherent cells, remove culture medium and wash cells once with PBS. For suspension cells, centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant, and wash once with PBS. Phenol red or serum may interfere with detection; a vacuum pump is recommended for removing medium and PBS. Washing may be omitted if residual liquid can be thoroughly removed. One wash generally reduces background fluorescence, but good staining can be achieved without washing.(3) Staining. Add an appropriate volume of Fluo‑3 Staining Solution: typically 100 μL per well for 96‑well plates, 250 μL for 24‑well plates, 500 μL for 12‑well plates, and 1 mL for 6‑well plates. Incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted between 10 and 60 min.Note 1: For initial experiments with uncertain temperature and time, test incubation at 37 ℃ for 30 min first. Shorten time or lower temperature if excessive cell death occurs; extend time if fluorescence intensity is too weak.Note 2: Cells may be washed 1–3 times with PBS or HBSS after incubation.(4) Detection. After incubation, observe staining under a fluorescence microscope (Fluo‑3 AM emits green fluorescence, Ex/Em = 490/525 nm).3. Detection by Flow Cytometry(1) Cell preparation. Harvest adherent cells by trypsin digestion, resuspend in medium, and wash once with PBS. For suspension cells, centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant, and wash once with PBS. Recommended cell amount per sample is 10⁶ cells.(2) Staining. To the pellet of 10⁶ cells from the previous step, add 1 mL Fluo‑3 Staining Solution and resuspend into a single‑cell suspension. Incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted between 10 and 60 min.Note 1: For initial experiments with uncertain temperature and time, test incubation at 37 ℃ for 30 min first. Shorten time or lower temperature if excessive cell death occurs; extend time if fluorescence intensity is too weak.Note 2: Cells may be washed 1–3 times with PBS or HBSS after incubation.(3) Detection. After incubation, analyze directly by flow cytometry, or centrifuge at 250–1000×g for 5 min at room temperature to pellet cells, remove liquid, resuspend in 0.5 mL Assay Buffer per sample, and analyze (Fluo‑3 AM emits green fluorescence, Ex/Em = 490/525 nm).Note 1: Use unstained cells in Assay Buffer as the negative control for flow cytometry setup.Note 2: As flow cytometry is highly sensitive, the probe concentration used may be lower than for microscopy; adjust the dilution of Fluo‑3 AM according to cell type and actual staining.4. Detection by Fluorescence Microplate Reader(1) Seeding and culture. Seed cells in black 96‑well plates. Cell number per well should be controlled within 100–10,000, typically 2000–5000. Treat cells as required by experimental design.(2) Washing (optional). For adherent cells, remove culture medium and wash once with PBS. For suspension cells, centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant, and wash once with PBS. Phenol red or serum may interfere with detection; a vacuum pump is recommended for removing medium and PBS. Washing may be omitted if residual liquid can be thoroughly removed. One wash generally reduces background fluorescence, but good staining can be achieved without washing.(3) Staining. Add an appropriate volume of Fluo‑3 Staining Solution, typically 100 μL per well in 96‑well plates. Incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted between 10 and 60 min.Note 1: For initial experiments with uncertain temperature and time, test incubation at 37 ℃ for 30 min first. Shorten time or lower temperature if excessive cell death occurs; extend time if fluorescence intensity is too weak.Note 2: Cells may be washed 1–3 times with PBS or HBSS after incubation.(4) Detection. After incubation, measure using a fluorescence microplate reader (Fluo‑3 AM emits green fluorescence, Ex/Em = 490/525 nm).The effect of drug stimulation can be determined by comparing RFU (Relative Fluorescence Units) between control and treated groups.References1. Clapham DE. Cell. 2007. 131(6):1047–58.2. Wu N, Nishioka WK, Derecki NC, Maher MP. Sci Rep. 2019. 9(1):12692.3. Hagen BM, Boyman L, Kao JP, Lederer WJ. Cell Calcium. 2012. 52(2):170–81.

Grade: BioReagent;Biological Stain;for Fluorescence analysis;for Microscopy

Storage Temperature: Protected from light;Store at -20°C

Shipped in: Ice chest + Ice pads