Calcium Kit - Fura 2CS23 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 the response to excitation at 380 nm decreases. The fluorescence intensity ratio (R = Fex340/Fex380) permits 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 – Fura 2 contains Fura 2-AM, together with the buffers required for the measurement of intracellular Ca²⁺ concentration. 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. Real-time measurements are possible using various fluorescence plate readers that are equipped with injectors for high-throughput screening. 

Fura 2-AM

Fura 2-AM    50 μg × 10

Dimethylsulfoxide (DMSO)    2 ml × 1

Recording Medium (2X)    100 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

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

Store at −20°C.

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

- Prepare the Fura 2-AM/DMSO solution, Loading Buffer and Recording Medium immediately before use.
- Use the Fura 2-AM/DMSO solution and Loading Buffer on a single occasion, because of the instability of the Fura2-AM.
- Use a plate reader capable of excitation at 340 and 380 nm. 
- If an instrument capable of dual-wavelength measurements or excitation at 340 and 380 nm is unavailable, we recommend using the Calcium Kit – Fluo 4 (code: CS22).
- 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, culture approximately 15,000–40,000 cells/well in a 96-well plate or 5,000–15,000 cells/well in a 384-well plate.
• Recommended culture volume: 100 μl/well (for a 96-well plate) or 25 μl/well (for a 384-well plate).

 

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

1. Add 50 μl of DMSO to one vial of Fura 2-AM (50 μg). Mix well until completely it dissolved.
2. Transfer 5 ml of Recording Medium (2X) to a 10 ml measuring flask. Add the desired amounts^※ 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 (the 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 thoroughly by ultrasonication, and use this as the Loading Buffer.

 

 3. Preparation of Recording Medium (1X)

1. Transfer 5 ml of Recording Medium (2X) to a 10 ml measuring flask. Add an appropriate amount^※ of 250 mmol/l Probenecid according to  the measurement conditions. Add distilled water to bring the final volume to 10 ml and mix thoroughly (the kit is pre-formulated to maintain a pH of 7.4).

• Recommended concentration: Probenecid 1.25 mmol/l.

When preparing 10 ml of Recording Medium (1X), the relationship between the final concentration of Probenecid during the assay and the amount added is as follows.

Amount Added and the final concentration of 250 mmol/l Probenecid solution
Amount Added (μl)	20	30	40	50	60
Final concentration (mmol/l)	0.50	0.75	1.00	1.25	1.50

 

2. Warm the solution in a 37°C incubator before use.

 

4. Loading Fura 2-AM into Cells

1. Remove the medium carefully and add 100 μl/well (for a 96-well plate) or 25 μl/well (for a 384-well plate) Loading Buffer. If necessary, wash the cells with PBS prewarmed to 37°C before adding the Loading Buffer.
2. Incubate at 37°C for 1 hour.
3. Carefully remove the Loading Buffer and add prewarmed Recording Medium (1X): 100 μl/well (for a 96-well plate) or 25 μl/well (for a 384-well plate). If needed, wash the cells with PBS before adding the Recording Medium.
4. Measure the changes in fluorescence intensity after the addition of compound using a fluorescence plate reader.
   （ λ_ex = 340 nm/380 nm, λ_em = 510 nm）