Calcium Kit II - Fura 2CS33 Product Manual

Fura 2-AM is widely used for the measurement of intracellular Ca²⁺ concentration. As the intracellular calcium concentration increases, the fluorescence intensity in response to excitation at 340 nm increases, whereas that in response to excitation at 380 nm decreases. The fluorescence intensity ratio (R = Fex340/Fex380) can be used for the quantitative evaluation of intracellular Ca²⁺ concentration, because it avoids the influence of factors such as the dye concentration, light source intensity, and cell size. 

The Calcium Kit II – Fura 2 contains Fura 2-AM and the buffers and components required for the measurement of intracellular Ca²⁺. The concentrations of Pluronic® F-127 or Cremophor® EL (solubilization aids for Fura 2-AM) and probenecid (an anion transporter inhibitor) can be adjusted according to the cell type and the compounds to be added, and these reagents can be added directly to microplates containing cultured cells. 

The kit contains reagents that quench the fluorescence of extracellular Fura 2, eliminating the need for cell-washing steps. Thus, it permits the rapid processing of many samples and is suitable for high-throughput screening. However, the quenchers may interfere with the assay when certain cell types and compounds are used. Under these circumstances, we recommend using the Calcium Kit – Fura 2 kit (code: CS23), which incorporates washing steps. 

The kit contains reagent volumes sufficient for 10 × 96-well plates. For the measurements, use clear-bottomed plates and a fluorescence plate reader that is capable of excitation and detection from below. 

Fura 2-AM

Fura 2-AM    50 μg × 10

Dimethylsulfoxide (DMSO)    2 ml × 1

Hanks’ HEPES Buffer (10X)    6 ml × 1

250 mmol/l Probenecid    1.3 ml × 1

5% Pluronic® F-127    2.5 ml × 1

5% Cremophor® EL    2.5 ml × 1

Quenching Buffer    55 ml × 1

• The volumes are sufficient for the assay of 10 x 96-well plates.

Store at −20°C. 

- 10 ml measuring flask
- Fluorescence microplate reader with a reagent injector
- 96 or 384-well Microplate
- Incubator (37°C)
- Micropipettes
- 20–200 μl Multichannel pipette

- Prepare the Loading Buffer immediately before use.
- Use the Fura2-AM/DMSO solution and Loading Buffer on a single occasion, because of the instability of the Fura2-AM. 
- Slight coloration of the Quenching Buffer bottle may be observed, but this does not affect the quality of the product. 
- Precipitation may occur in the Quenching Buffer. If so, warm this at 40°C to dissolve the solid material before use. 
- This product contains glass containers. Wear protective gloves and handle with care.

1. Cell Culture

Dispense the cell suspension into each well of a microplate and incubate overnight at 37°C in an incubator equilibrated with 95% air and 5% CO₂.

• For adherent cells: 15,000 cells/well (96-well) or 5,000 cells/well (384-well).
• For suspension cells: 100,000 cells/well (96-well) or 25,000 cells/well (384-well).
• Recommended culture volume: 100 μl/well (96-well), 25 μl/well (384-well).

 

2. Preparation of Loading Buffer (for one 96-well plate)

1. Add 50 μl of DMSO to one vial of Fura 2-AM (50 μg) and dissolve it completely.
2. Transfer 5 ml of Quenching Buffer to a 10 ml measuring flask. Add 500 μl of Hanks’ HEPES Buffer (10X), the desired amount^※ of 5% Pluronic® F-127 (or 5% Cremophor® EL), and 250 mmol/l Probenecid according to the measurement conditions. Add distilled water to bring the final volume to 10 ml and mix thoroughly (this kit is pre-formulated to maintain a pH of 7.4).

• Recommended concentrations: Probenecid 1.25 mmol/l; Pluronic® F‑127 0.04%.

When preparing 10 ml of Loading Buffer, the relationships of the final concentrations of Probenecid and Pluronic® F-127 (or Cremophor® EL) during the assay with the amounts added are as follows.

 

3. Add 50 μl of the Fura 2-AM/DMSO solution, mix well, and use as Loading Buffer.

 

3. Loading Fura 2-AM into Cells

1. Without removing the culture medium, add an equal volume of Loading Buffer directly to each well (100 μl/well for 96-well plates and, 25 μl/well for 384-well plates).
2. Incubate at 37°C for 1 hour.
3. Measure the changes in fluorescence after the addition of compound using a fluorescence plate reader.
   (λ_ex = 340/380 nm, λ_em = 510 nm; no washing required.)