Calcium Kit - Fluo 4CS22 Product Manual

The Calcium Kit – Fluo 4 is a screening kit that contains Fluo 4-AM, a reagent for measuring intracellular Ca²⁺ concentration, together with the required buffers and other components. The concentrations of Pluronic® F-127 or Cremophor® EL (solubilization aids for Fluo 4-AM) and probenecid (an inhibitor of anion transporters) can be adjusted according to the cell type and the compounds to be added. Various fluorescence plate readers equipped with injectors for high-throughput screening can be used to measure the real-time changes in intracellular Ca²⁺ concentration after the addition of a compound. 

Fluo 4-AM

Fluo 4-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 (37°C)
- Micropipettes
- 20–200 μl Multichannel pipette

- Preparethe  Fluo 4-AM/DMSO solution, Loading Buffer, and Recording Medium immediately before use.
- Use the Fluo 4-AM/DMSO solution and Loading Buffer on a single occasion, because of the instability of the Fluo 4-AM. 
- For more accurate measurement of the intracellular calcium ion concentration, we recommend using the Calcium Kit – Fura 2 (code: CS23). However, an instrument capable of dual-wavelength excitation at 340 nm and 380 nm is required. 

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 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 (96-well) or 25 μl/well (384-well).

 

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

1. Add 50 μl of DMSO to one vial of Fluo 4-AM (50 μg). Mix well until dissolved it completely .
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 (this kit is pre-formulated to maintain pH 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 concentration 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 Fluo 4-AM/DMSO solution, mix thoroughly by ultrasonication, and use this solution 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. Incubate at 37°C before use.

 

4. Loading Fluo 4-AM into Cells

1. Remove the medium carefully and add Loading Buffer: 100 μl/well (96-well plate) or 25 μl/well (384-well plate). 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 the prewarmed Recording Medium (1X): 100 μl/well (96-well plate) or 25 μl/well (384-well plate). If necessary, wash the cells with PBS prewarmed to 37°C before adding the Loading Buffer.
4. Measure the changes in fluorescence intensity after the addition of compounds using a fluorescence plate reader.
   （λ_ex = 480–500 nm, λ_em = 518 nm）