alamarBlue™ Standard Cell Viability Reagent, 100 mL - FAQs

可能的原因之一是您的alamarBlue试剂中的染料已经沉淀，导致染料浓度变化。这种情况下，应将试剂加热至37°C，然后振荡或漩涡，确保所有的组分都溶于溶液中。另一个原因是移液问题。请确保你的移液器经过校准且吸液前枪头固定牢固。 

请缩短孵育时间和/或减少实验所用的细胞数量。 

我们建议增加细胞在alamarBlue试剂下的孵育时间，更改仪器的增益或电压设置，检查仪器的滤光片/波长设置。务必在实验设计时安排一个阳性对照（未处理的活细胞）组。  

试剂遇光可能会分解。务必在避光条件下储存alamarBlue试剂，且不要长时间将试剂直接暴露于光照下。 

由于指示剂是一种多组分溶液，我们建议将冷冻的alamarBlue试剂加热至37°C，并振荡或漩涡，确保所有的组分都溶于溶液中。 

室温条件下（~22°C），试剂至多可稳定保存12个月。 

可以。alamarBlue试剂经过多次冻/融仍可保持稳定。使用之前，务必在37°C下水浴加热并混合均匀，确保溶液均质。 

alamarBlue试剂和PrestoBlue试剂含有的刃天青包含在一种专有的稳定配方中，能够提供了一种便捷的“混合、接种和读数”实验方案。PrestoBlue试剂是alamarBlue试剂的改进配方允许更快速的染色（典型的10分钟相比于1-4小时获得相似的信号和灵敏度）。C12-刃天青是刃天青的衍生物，其拥有良好的细胞滞留性，因此适用于伴有活性指示剂和其他生物标记的流式细胞分析仪多重检测分析。

已被验证过的alamarBlue 细胞活力试剂，PrestoBlue细胞活力试剂, 和CyQUANT 细胞增殖实验试剂盒（货号C7026）可用于AlgiMatrix 3D培养体系，同时也可以与其他3D培养体系一同使用。更多细节，请参考这个海报（https://aacrjournals.org/cancerres/article/70/8_Supplement/3234/563757/Abstract-3234-AlgiMatrixT-3-D-cell-culture-system）和手册（https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/3-d-cell-culture-protocol/algimatrix-viability-using-cyquant.html）。 

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。

胎牛血清（FBS）和牛血清蛋白（BSA）会引起一些荧光淬灭。我们建议在对照组中使用同样浓度血清来体现这种淬灭。假定培养基不含还原剂，其他培养基组分不干扰实验。如果生长培养基的pH没有显著变化，则其中的酚红基本不存在干扰。在中性pH或其附近，酚红的存在仅仅会提高大约0.03单位的值。理论上，实验应该在没有酚红的培养基中进行。 

您可能需要根据每种类型细胞决定alamarBlue实验铺板密度和孵育时间，使用可以使实验处于线性范围内的条件。

铺板密度：

当细胞处于指数生长期时，alamarBlue试剂测定的细胞增殖最准确。一旦细胞密度太高，细胞增殖就会减少，alamarBlue试剂的还原低于期望值。低细胞密度时，低的生长速率会导致alamarBlue的还原不显著。通常建议处于对数生长期的动物细胞密度为1 x 10E4细胞/mL。然而，由于细胞增殖速率不同，不可能为所有的实验推荐一个细胞密度。取而代之的，我们建议进行一个对照实验来确定您实验的最佳细胞密度。产品手册中提供了详细的方案。

孵育时间：

优化alamarBlue试剂孵育时间同样重要。将细胞在alamarBlue试剂中37°C或者在种群/细胞类型最佳的温度条件下培养1-4小时。为了更加灵敏地检测低密度细胞数量，增加孵育时间到24小时。如果您计划使用长的孵育时间（过夜），在添加试剂和接种时确保维持无菌条件避免微生物污染物。由于微生物污染物同样会还原alamarBlue试剂，污染的培养基会产生错误的结果。产品手册中提供了详细的方案。

alamarBlue试剂包含了专有的缓冲液试剂，可以维持溶液中氧化或还原形式的alamarBlue试剂和其他组分的稳定性和可溶性。过长时间孵育细胞，缓冲液能力会变低，试剂的稳定性和可溶性都会受到影响，导致信号丢失。

另外，如果实验的长度长于alamarBlue最佳的培养时间，我们建议使用终点法检测。这种类型的实验对于超过几天、几周和几个月的细胞增殖研究特别有用。alamarBlue试剂能用于长期的反复测定的细胞增殖研究。对于此类研究，在终点法检测之前，我们建议每个时间点取得小份的细胞培养基/悬浮液加入alamarBlue试剂进行孵育。

我们建议设置合适的实验对照。为了最小化实验误差，我们推荐对实验和无细胞对照设置最少4到8个重复，从而测量数值。一些建议如下：

•无细胞对照：只有培养基的孔可用于测定背景荧光值，并可从实验组孔数值中将其减去。我们建议在对照组中采用相同浓度的血清。酚红可能会干扰实验：理论上实验应该在不含酚红的培养基中进行。
•无细胞+实验化合物对照：仅含培养基并添加化合物来评估实验中使用药品/化合物导致的潜在的背景荧光或吸光度。
•未经处理细胞对照：如果您用化合物处理细胞，我们建议您设置未处理细胞的孔。
•溶剂对照：如果您用有机溶剂例如DMSO、乙醇等溶解的化合物处理细胞，我们推荐您设置仅添加了有机溶剂和细胞的孔（与添加到实验样品中的体积相同，无化合物）：这个对照得到的结果应该与未处理细胞对照相似。如果这些样品得到的结果与未处理的对照不同，此样品可能对溶剂敏感。

•荧光：使用530–570 nm（激发峰为570 nm）间的激发波长进行荧光读数。使用580–610 nm（发射峰为585 nm）间的发射波长进行荧光读数。
•吸光度：在570 nm处读取alamarBlue试剂的吸光值，使用600 nm作为参考波长（以600 nm的值进行归一化）。

注：荧光检测更加敏感。当无法使用荧光仪时，可以测量alamarBlue试剂的吸光值。对于大多数动物细胞样本，实验平板或试管可以用锡箔纸或塑料薄膜包裹（防止蒸发），4°C贮存，1-3天内读值不会影响荧光或吸光值。这可能不适用于细菌、藻类、原生动物或真菌样品。

alamarBlue细胞活力试剂对光敏感，需要避光储存。产品可以在室温下储存12个月。保质期见产品标签。如果要储存超过12个月，在2-8°C条件下储存可将储存时间增加至20个月。alamarBlue试剂可以在低于–70°C条件下长久储存。因为此指示剂是一个多组分溶液，我们建议冷冻的alamarBlue试剂加热至37°C，摇匀确保所有的组分完全溶解。

alamarBlue试剂适用于多种不同种类细胞：哺乳类动物、鸟类、两栖类动物、鱼类、硅藻属、细菌、植物细胞、真菌等，以及其他细胞样品，例如无限增殖培养细胞、原代培养细胞、癌细胞、干细胞等。同时，该试剂还可用于测定组织样品活性，例如心脏瓣膜和AlgiMatrix 3D矩阵中培养的细胞。这类应用有许多参考文献。您可以通过搜索alamarBlue和您的目的细胞类型来找到它们。例如，使用谷歌学术，使用搜索项‘alamarBlue and cancer cells’ 或 ‘alamarBlue and tissue’。

尽管大部分文献报道alamarBlue试剂中的活性成分刃天青对细胞无毒，但其他报道表明细胞活力会受到影响，且取决于刃天青孵育的时间和孵育浓度。 

alamarBlue细胞活力试剂是一个可靠的细胞活力和增殖的指示剂，包含有效成分刃天青，一种无毒、细胞通透型不带荧光的蓝色指示染料。健康的细胞在其细胞质中具有还原活力。alamarBlue扩散进入细胞后，活细胞中潜在的天然还原力将刃天青转变成亮红色的细胞膜通透型荧光染料试卤灵。刃天青通过脱氧被还原。

  活细胞连续不断地将刃天青转变为试卤灵，从而对活性、增殖、细胞毒性进行数量测定。净结果是可以测量的颜色和荧光强度的变化。荧光或吸光值与活细胞的数量成正比，反映了细胞代谢活性。相比于健康的细胞，损伤和死亡的细胞代谢活性低因此产生强度信号相对较低。

可采用PrestoBlue细胞活力染色剂和CyQUANT细胞增殖检测试剂盒。我们还提供神经突生长染色试剂盒（货号A15001）。更多有关我们的神经元细胞健康检测试剂的信息请见此处（https://www.thermofisher.com/us/en/home/life-science/cell-analysis/neuroscience/neuronal-cell-health-assays.html）。

除了Invitrogen alamarBlue 细胞活力检测试剂（Cell Viability Reagent）之外，AlgiMatrix 基质还能够与Invitrogen PrestoBlue 细胞活力检测试剂，Promega  Cell Titer-Glo 试剂或Invitrogen CyQuant  Direct Cell Proliferation Assay试剂盒相兼容。具体实验方案与2D细胞培养系统几乎相同，但需要加入纯AlgiMatrix 基质作为空白对照组。

Invitrogen alamarBlue 检测是一项通过监测还原能力来监控细胞健康和细胞功能情况的有用的生物分析法。

This is true for MTT (as well as XTT, AlamarBlue Cell Viability Reagent, and PrestoBlue Cell Viability Reagent). CyQUANT Direct will also only count live cells, but for a different reason. The dye in it is a green-fluorescent nucleic acid stain, which will bind to DNA in all cells, live or dead, without the need for cellular metabolism. However, there is a non-cell-permeable quenching reagent in the kit which will both quench extracellular fluorescence (and thus this is a no-wash assay) AND will quench the fluorescence in dead cells (but not live cells).

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This is true for MTT (as well as XTT, AlamarBlue Cell Viability Reagent, and PrestoBlue Cell Viability Reagent). CyQUANT Direct will also only count live cells, but for a different reason. The dye is a green-fluorescent nucleic acid stain, which will bind to DNA in all cells, live or dead, without the need for cellular metabolism. However, there is a cell impermeant quenching reagent in the kit which will quench both extracellular fluorescence (and thus this is a no-wash assay) and the fluorescence in dead cells (but not live cells).

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alamarBlue HS Cell Viability Reagent provides a lower background, suitable for analyzing a lower number of cells per well. Also, the removal of impurities in the 'HS' reagent limits any artifacts that may be critical to your cells' viability.

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Yes and in addition, alamarBlue HS Cell Viability Reagent has a higher purity which results in a lower background.

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Both products utilize resazurin as the indicator dye. alamarBlue HS Cell Viability Reagent undergoes a purification process that removes any residual resorufin that may be present from the standard manufacturing process for resazurin. This purification results in a product that has very low background. alamarBlue HS Cell Viability Reagent may be used on samples with as few as 20 cells/well, compared to a minimum of 50 cells/well for the original alamarBlue Cell Viability Reagent.
Note: Minimum cell number/well is also dependent upon reducing activity of the cell type. Results may vary.

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One reason could be that the dye in your alamarBlue reagent has precipitated, resulting in varying dye concentration. In such cases, the reagent should be warmed to 37 degrees C and shaken or swirled to ensure that all components are completely in solution. Another cause can be pipetting issues. Ensure that your pipettor has been calibrated and the pipette tips are securely anchored prior to pipetting.

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Decrease the incubation time and/or reduce the number of cells used in the experiment.

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We recommend increasing the incubation time of cells with alamarBlue reagent, changing the instrument's gain or voltage setting, and checking the instrument filter/wavelength settings. Make sure to have positive controls (untreated, living cells) in the experimental design as a control.

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The reagent may be breaking down due to exposure to light. Be sure to store alamarBlue reagent in the dark and do not expose the reagent to direct light for long periods of time.

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Because the indicator is a multicomponent solution, we recommend that frozen alamarBlue reagent be warmed to 37 degrees C and shaken or swirled to ensure that all components are completely in solution.

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The reagent is stable for up to 12 months when stored at room temperature (approximately 22 degrees C).

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Yes. alamarBlue reagent is stable to multiple freeze/thaw cycles. Be sure to warm the reagent in a 37 degrees C water bath and mix it well to ensure a homogenous solution before use.

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alamarBlue reagent and PrestoBlue reagent contain resazurin in a proprietary stabilizing formulation that allows for a convenient “mix, incubate, and read” protocol. PrestoBlue reagent is an improvement in the formulation of alamarBlue reagent that allows for much faster staining (typically 10 minutes vs. 1-4 hours to obtain a similar signal and sensitivity). C12-resazurin is a derivative of resazurin that has better cellular retention and thus allows for analysis on a flow cytometer and multiplexing with viability indicators and other biomarkers.

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alamarBlue Cell Viability Reagent, PrestoBlue Cell Viability Reagent, and CyQUANT Cell Proliferation Assay Kit (Cat. No. C7026) have been validated for use with the AlgiMatrix 3D Culture System and should also work with other 3D culture systems. For further details, please refer to this poster (https://aacrjournals.org/cancerres/article/70/8_Supplement/3234/563757/Abstract-3234-AlgiMatrixT-3-D-cell-culture-system) and protocol (http://www.thermofisher.com/us/en/home/references/protocols/cell-culture/3-d-cell-culture-protocol/algimatrix-viability-using-cyquant.html).

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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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Fetal bovine serum (FBS) and bovine serum albumin (BSA) cause some quenching of fluorescence. We recommend using the same serum concentration in controls to account for this quenching. Assuming the media does not contain reducing agents, other media components do not interfere with the assay. There is no interference from the presence of phenol red in the growth medium if the pH has not changed significantly. At or near neutral pH, the presence of phenol red merely shifts the values approximately 0.03 units higher. Ideally, the assay should be done in phenol red-free medium.

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You may need to determine the plating density and incubation time for the alamarBlue assay for each cell type and use conditions such that the assay is in the linear range.

Plating Density:
alamarBlue reagent measures cell proliferation most accurately when the cells are in the exponential growth phase. If the cell density is too high, cell proliferation will decrease, giving less reduction of the alamarBlue reagent than would have been expected. At low cell density, the slower growth rate could result in insignificant alamarBlue reduction. A cell density of 1 x 10E4 cells/mL is generally recommended for animal cells, with cells in the exponential phase. However, since cells vary in their proliferation rate, it is impossible to recommend a cell density which is suitable for all experiments. Instead we recommend performing a control experiment to determine the optimum cell density for your studies. A detailed protocol is provided in the product manual.

Incubation Time:
It is equally important to optimize the incubation time for the alamarBlue reagent. Incubate the cells with alamarBlue reagent for 1-4 hours at 37 degrees C or, at an optimal temperature for the species/cell type. For more sensitive detection with low cell numbers, increase the incubation time for up to 24 hours. If you plan to use longer incubation time (overnight), be sure to maintain sterile conditions during reagent addition and incubation to avoid microbial contaminants. Contaminated cultures will yield erroneous results as microbial contaminants also reduce alamarBlue reagent. A detailed protocol is provided in the product manual.

alamarBlue reagent contains proprietary buffering agents which maintain the stability and solubility of both the oxidized and reduced forms of the alamarBlue reagent and other components in the solution. Over extended incubation times with cells, the buffering capacity may be altered to such an extent that reagent solubility and stability may be affected, resulting in a loss of signal.

Alternatively, if the length of the experiment is longer than the optimal alamarBlue incubation time, we suggest that an endpoint test is used. This type of experiment is particularly useful for cell proliferation studies over days, weeks and months. alamarBlue reagent can be used for long-term cell proliferation studies where measurements are taken repeatedly. For these types of studies, we recommend that aliquots of the cell medium/ suspension are taken at each time point during incubation with alamarBlue reagent prior to an endpoint.

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We recommend including appropriate assay controls. To minimize experimental errors, we recommend making measurements from a minimum of 4-8 replicates of experimental and no-cell control samples. Some suggestions are provided below:

No-cell controls: Wells that contain only culture media so that the background fluorescence can be determined and subtracted from experimental wells. We recommend using the same serum concentration in controls. Phenol red may interfere with the assay; ideally the assay should be performed using Phenol red-free media.
No-cell + experimental compound controls: Wells that contain only cell culture media and added compound to assess potential background fluorescence or absorbance caused by the drug/chemical used in the experiment.
Untreated-cell controls: If you're treating cells with a compound, we recommend that you plate wells of untreated cells.
Solvent controls: If you're treating cells with a compound dissolved in a solvent such a DMSO, ethanol, etc., we recommend that you plate wells of cells with added solvent only (same volume added to experimental samples, no compound); this control should provide similar results as untreated cell controls. If results from these samples are different from untreated cell controls, the sample may be sensitive to the solvent.

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Fluorescence: Read fluorescence using an excitation wavelength between 530-570 nm (peak excitation is 570 nm). Read fluorescence emission between 580-610 nm (peak emission is 585 nm).
Absorbance: Monitor the absorbance of alamarBlue reagent at 570 nm, using 600 nm as a reference wavelength (normalized to the 600 nm value).
Note: Fluorescence detection is more sensitive. When fluorescence instrumentation is unavailable, monitor the absorbance of alamarBlue reagent. With most animal cells, assay plates or tubes can be wrapped in foil or plastic wrap (to prevent evaporation), stored at 4 degrees C, and read within 1-3 days without affecting the fluorescence or absorbance values. This may not hold true for bacterial, algal, protozoan, or fungal samples.

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alamarBlue Cell Viability Reagent compound is light sensitive and should be stored in the dark. The product may be stored for 12 months at room temperature. The expiration date is given on the product label. If shelf life beyond 12 months is desired, storing at 2-8 degrees C increases shelf life to 20 months. alamarBlue reagent may also be frozen at greater than -70 degrees C indefinitely. Because the indicator is a multicomponent solution, we recommend that frozen alamarBlue reagent be warmed to 37 degrees C and shaken to ensure all components are completely in solution.

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alamarBlue reagent has been used with many different cell species: mammalian, avian, amphibian, fish, diatoms, bacteria, plant cells, fungi and others-and cell samples, such as immortalized cell cultures, primary cell cultures, cancer cells, stem cells, etc. Also, alamarBlue reagent has been used to measure viability of tissue samples such as heart valves and cells cultured in 3D matrices such as AlgiMatrix matrix. There are many references available for this application. You can find them by searching for alamarBlue and your cell type of interest. For example, using Google Scholar, use the search term ‘alamarBlue and cancer cells' or ‘alamarBlue and tissue'.

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While most reports in the literature suggest that solutions containing resazurin, the active ingredient in alamarBlue reagent, are not toxic to cells, other reports clearly show that cell viability is affected depending on the length of exposure and concentration of resazurin to which they are subjected.

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alamarBlue Cell Viability Reagent is a proven cell viability and proliferation indicator. It contains the active ingredient, resazurin, a non-toxic, cell permeable, non-fluorescent blue indicator dye. Healthy living cells maintain a reducing state within their cytosol. Upon diffusion into cells, the natural reducing potential of living cells converts resazurin to the very bright red, cell permeable fluorescent dye, resorufin. Resazurin is reduced by the removal of oxygen.

Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability, proliferation, and cytotoxicity. The net result is measurable change in the color and fluorescence intensity. The amount of fluorescence or absorbance is proportional to the number of living cells and corresponds to the cells' metabolic activity. Damaged and nonviable cells have lower metabolic activity and thus generate a proportionally lower signal than healthy cells.

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PrestoBlue Cell Viability Stain and CyQUANT Cell Proliferation Assay Kit can be used. We also offer a Neurite Outgrowth Staining Kit (Cat. No. A15001). More information about our different assays for neuronal cell health can be found here (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/neuroscience/neuronal-cell-health-assays.html).

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Other than the Invitrogen alamarBlue Cell Viability Reagent, AlgiMatrix Matrix is also compatible with Invitrogen PrestoBlue Cell Viability Reagent and Invitrogen CyQuant Direct Cell Proliferation Assay. The protocol is almost the same as the 2D cell culture, but it needs the blank AlgiMatrix Matrix as a blank control.

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The Invitrogen alamarBlue reagent assay is a useful biological assay that monitors the health and function of cells by monitoring their reducing functions.