Indo-1, AM, cell permeant - Special Packaging - FAQs

Q: 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 K d 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). Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center .

查看更多产品信息 Indo-1, AM, cell permeant - Special Packaging - FAQs (I1223)

4 个常见问题解答

使用流式细胞仪时，可以使用什么指示染料来测定钙离子流？

Indo-1，AM流式细胞仪的首选染料。它更适用于单激光激发（通常使用351-364 nm的氩离子激光的谱线），可监视两个发射。Indo-1的最大发射从在无Ca2+培养基中的〜475 nm变为Ca2+饱和的〜400 nm。Indo-1，AM是特别适用于多色荧光应用。

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 K_d 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).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

What indicator dye can I use to measure calcium flux by flow cytometry?

Indo-1, AM is the preferred stain for flow cytometry, where it is more practical to use a single laser for excitation, usually the 351–364 nm spectral lines of the argon-ion laser, and monitor two emissions. The emission maximum of Indo-1 shifts from ~475 nm in Ca2+-free medium to ~400 nm when the dye is saturated with Ca2+. Indo-1, AM is particularly suited for multicolor fluorescence applications.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.