Calcium Kit II - Fluo 4CS32 Product Manual

The Calcium Kit II – Fluo 4 is a kit that contains Fluo 4-AM, a reagent used to measure the intracellular Ca²⁺ concentration, together with the buffers and components required for its measurement. The concentrations of Pluronic® F-127 or Cremophor® EL (solubilization aids for Fluo 4-AM) and probenecid (an anion transporter inhibitor) can be adjusted according to the cell type and the compounds to be added, and can be added directly to a microplate containing cultured cells. The kit includes reagents that quench the fluorescence of extracellular Fluo 4, eliminating the need for conventional cell-washing procedures. Thus, this kit permits the rapid processing of numerous 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 – Fluo 4 (code: CS22), which incorporates washing steps. This kit contains reagents sufficient for 10 × 96-well plates. For the measurements, use clear-bottomed plates and a microplate reader that is capable of excitation and fluorescence detection from below. 

Fluo 4-AM

Fluo 4-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 Fluo 4-AM/DMSO solution and Loading Buffer immediately before use.
- Use the Fluo 4-AM/DMSO solution and Loading Buffer on a single occasion, because of the instability of Fluo4-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, culture approximately 15,000 cells/well (for a 96-well plate) or 5,000 cells/well (for a 384-well plate).
• For suspension cells, culture approximately 100,000 cells/well (for a 96-well plate) or 25,000 cells/well (for a 384-well plate).
• Recommended culture volumes: 100 μl/well (for a 96-well plate), 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 Fluo 4-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 Fluo 4-AM/DMSO solution, mix well until the solid material is dissolved, and use this as the Loading Buffer. 

 

3. Loading Fluo 4-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 fluorescence changes after the addition of compound using a fluorescence plate reader.
   (λ_ex = 480–500 nm, λ_em = 518 nm; no cell washing is required.)