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Molecular Probes™ Fluo-4 Direct™ Calcium Assay Kit

Catalog No. F10472
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1 x 100 mL
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Description

Fluo-4 Direct™ Calcium Assay Kit

The Fluo-4 Direct™ Calcium Assay Kit was formulated to provide a homogeneous fluorescent calcium assay that:

  1. Easily loads into cells
  2. Achieves a large assay window and
  3. Suppresses background fluorescence generated from the calcium indicator in complete media with little-to-no impact on the specific cellular fluorescence

This advanced formulation allows the assay to be run in a simple 'addition only' format in the presence of serum-containing media. Fluo-4 Direct™ is the third addition to the Fluo-4 family of calcium detection reagents. Fluo-4 AM and Fluo-4 NW both require media removal before assay detection, but Fluo-4 NW adds convenience by including the PowerLoad™ reagent for ease in cell loading. The Fluo-4 Direct™ Calcium Assay Kit is similar to Fluo-4 NW in that it is formulated with PowerLoad™ for ease in cell loading, but unique in that it can be used in the presence of complete culture media and will efficiently suppress background fluorescence without sacrificing the specific cellular fluorescence generated in the assay.

Specifications

Color Green
Content And Storage
  • 100 ml Fluo-4 Direct™ Calcium Assay Reagent (Component A)
  • 2 × 77 mg Probenecid (Component B)
  • 200 ml Fluo-4 Direct™ Calcium Assay Buffer (Component C)
  • Store at ≤-20°C. Dessicate and protect from light.
Detection Method Fluorescence
For Use With (Application) Calcium Assay
For Use With (Equipment) Confocal Microscope, Fluorescence Microscope, High Content Analysis Instrument, HTS Reader, Microplate Reader, Fluorescent Imager
Product Type Calcium Assay Kit
Dye Type Fluorescent Dye-Based
Emission Visible
Form Powder
Modification Chemical
Product Line Molecular Probes
Quantity 1 x 100 mL
Shipping Condition Room Temperature
Sub Cellular Localization Nucleus, Organelles, Cytoplasm
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Frequently Asked Questions (FAQs)

I am doing calcium flux imaging with your Fura-2 calibration kit, but am seeing a large variability in ratio in different places around the slide. I am correcting for uniform illumination, using the product as directed, and sealing the coverslip with nail polish.

The nail polish may be the problem. The Kd value (calcium sensitivity) changes depending upon the dye's environment. Nail polish has solvents that can leech under the coverslip and cause variability. We recommend either going without a sealing or sealing with melted paraffin painted on the coverslip edges with a cotton-tipped applicator (paraffin is hydrophobic and has no solvents).
I need to label cells with Fluo-4, AM, for a calcium flux assay. How long after labeling will the dye be retained?

After loading dye into the cells, intracellular esterases remove the 'AM' moiety from the dye. When the 'AM' group is removed, the dye is able to bind calcium and fluoresce. Since the dye is not covalently bound to any cellular components, it may be actively effluxed from the cell. The rate of efflux is dependent upon the inherent properties of the cell, culture conditions and other factors. The dye may be retained for hours, days or even weeks or lost in a matter of minutes. The use of Probenecid (Cat. No. P36400) limits loss by active efflux.

For Research Use Only. Not for use in diagnostic procedures.