Calcein AM, Cell-permeant Green Dye, 20 x 50 μg - FAQs

不可以，不可能测试相同群体细胞的活性超过几小时；您可以使用重复的样品。DNA结合的染料对细胞有毒；染色后细胞应尽快成像。钙黄绿素AM不能停留在细胞中，根据不同类型的细胞，在几分钟到若干小时内，其可能会被细胞主动地排出。钙黄绿素AM对细胞无毒，因此其可以重复添加到相同的样品中。您可以使用alamarBlue试剂或PrestoBlue试剂进行相同样品超过若干天的细胞增殖检测，因为这些染料对细胞是无毒性的。

一种可能性是染料之间的光谱渗漏。务必通过在绿光下成像红色细胞，在红光下成像绿色细胞的方式检查单色样品，对其他颜色使用最佳成像设置。如果您在对照中看到这些渗漏，那么就需要减少染料标记浓度以降低染料的亮度，或者选择那些光谱相距较远的染料。如果不是渗漏的问题，另一种可能性是细胞在染色后没有充分清洗，使得一些未结合的染料残留下来，这些染料进入后会标记其他细胞。延长清洗次数和时间应该会有帮助。

Calcein AM扩散到细胞中，“AM”部分由细胞酯酶裂解，随后染料分子的荧光信号可以在细胞质中被观测到，但其不结合到细胞组分。这意味着它们能够为“整个细胞”染色。这也意味着染料不能通过醛类固定剂进行交联，因此染料会在固定时丢失。此外，质膜的任何干扰（例如去垢剂或胰蛋白酶处理）都会导致染料从细胞中渗漏。

首先，确定您是在无血清条件下进行染色的。因为血清具有酯酶活性，会过早的裂解这些染料上的AM基团，进而阻止其进入细胞。染色之后即可将细胞移回含血清培养基中。除此之外，您可以尝试增加浓度和标记时间以获得更高的强度的信号。

钙黄绿素染料扩散到细胞中，“AM”部分由细胞酯酶切割，随后可以在细胞质中观察到而未结合任何胞内成分。这意味着它们标记了“整个细胞”。但是这也意味着它们可以通过正常细胞的外排机制被泵出。有时这一过程时间很短，尤其是显示出抗药性类型的细胞，除非外排被抑制（如使用丙磺舒抑制外排）。这也意味着染料不能被醛类固定剂所交联（不同于蛋白质结合CellTracker染料），因此染料会在固定时丢失。此外，质膜的任何干扰（例如去垢剂或胰蛋白酶处理）都会导致染料从细胞中渗漏。

Yes. This is commonly done with calcein AM or FDA (fluorescein diacetate). These dyes will not fluoresce until cleaved by esterases. After modification by esterases and at very high concentrations, they will self-quench. Upon disruption of the plasma membrane, or cell death, the dye will be released into the extracellular medium, and become unquenched. Concentration and incubation time must be optimized to obtain adequate quenching.

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Calcein, AM requires esterase cleavage of the acetoxymethyl (AM) ester to become fluorescent. Liposomes don't have esterases unless specifically constructed to include the enzyme. The water-soluble, non-AM form of calcein (Cat. No. C481), does not require esterase cleavage to be fluorescent.

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Calcein, AM and FDA (fluorescein diaceate) are examples of some dyes used for this application. Since these dyes are not incorporated or covalently attached to any cellular components, they may have a short retention time as some cell types may actively efflux the dye out of the cells. The CellTracker and CellTrace dyes include either a mild thiol-reactive chloromethyl group or amine-reactive succinnimidyl ester group to allow for covalent binding to cellular components, providing for better retention. As with any reagent, one should empirically determine retention times for the cell type used.

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This is not recommended. Neither Calcein nor ethidium homodimer-1 bind to any cellular components upon fixation. There is no guarantee that the dyes will be retained upon fixation or any subsequent wash steps. We recommend scoring for live and dead cells as soon as possible after staining.

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Calcein AM, a green dye, is typically used as a general cytoplasmic stain, but not recommended with GFP-positive cells. For GFP-expressing cells there are other colors available: Calcein Blue AM, Calcein Violet AM, and Calcein Red-Orange AM. The retention time of these dyes in live cells is dependent upon the inherent properties of the cell.

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Calcein, AM is a good choice for cell tracking and as a general cytoplasmic stain. However, it doesn't bind to anything and may be actively pumped out of the cells within a couple hours, which is likely what happened. The retention of Calcein within live cells is dependent upon the inherent properties of the cell type and culture conditions.

For long-term imaging, you may wish to consider a reactive cytoplasmic stains such as CFDA, SE or the CellTracker and CellTrace dyes.

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No, it is not possible to assay the viability of the same population of cells longer than a few hours; you will need to use replicate samples. DNA binding dyes are toxic to cells; stained cells should be imaged as soon as possible after staining. Calcein AM is not retained in cells and may be actively effluxed out in the range from minutes to several hours, dependent upon the cell type. Calcein AM is not toxic to cells, so it can be added repeatedly to the same samples. You can assay the proliferation of the same sample of cells over several days using alamarBlue reagent or PrestoBlue reagent, as these dyes are non-toxic to cells.

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One possibility is that there is spectral bleedthrough between the dyes. Be sure to check the single-color samples by imaging the red cells in green and imaging the green cells in red, using the optimal imaging settings for the other color. If you see bleedthrough with these controls, then you will have to reduce the dye label concentration to reduce the brightness of the dyes, or choose dyes that are farther apart spectrally. If the issue isn’t bleedthrough, another possibility is that the cells were not adequately washed after staining, allowing some unincorporated dye to remain and label the other cells after they were introduced. Extending washes and wash times should help with this.

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Calcein, AM diffuses into cells, the 'AM' moiety is cleaved by cellular esterases, and then the dye molecules are observed in the cytoplasm without binding to anything. This gives a 'whole cell' stain. It also means that the dyes are not crosslinked with aldehyde-based fixation and thus will be lost upon fixation. Additionally, any disruption of plasma membrane, such as with detergents or trypsinization, will lead to leakage of the dye from the cell.

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First, make sure you aren’t staining in the presence of serum, since serum can have esterase activity that can prematurely cleave the AM group on these dyes, preventing entry into cells. After staining, it’s okay to return the cells to medium containing serum. After this, you can try increasing the concentration and label time to get a higher intensity.

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Calcein dyes diffuse into cells, the 'AM' moiety is cleaved by cellular esterases and then are observed in the cytoplasm without binding to anything. This provides a 'whole cell' label. Calcein dyes may be pumped out by normal cellular efflux mechanisms, sometimes within a very short time, especially for cell types that may exhibit drug resistance, unless the efflux is inhibited (such as with probenecid). The dyes are not crosslinked with aldehyde-based fixation, unlike protein-binding CellTracker dyes, and thus will be lost upon fixation. Additionally, any disruption of plasma membrane, such as with detergents or trypsinization, will lead to leakage of the dyes from the cell.

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