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alamarBlue—Rapid & Accurate Cell Health Indicator
Invitrogen offers alamarBlue, an important reagent for evaluating cellular health. For researchers who need to assess whether cells have enough energy to proliferate, this reagent provides a nontoxic, scalable method. Ready-to-use alamarBlue is a more cost effective, easier, and safer way for you to get the results you need.
- Superior performance
- Fast, simple and scalable assay
- Economical and safe
Understand the advantages of alamarBlue and why this may be important to your research.
Superior Performance
- Quantitative—provides accurate measurement over time
- High sensitivity and linearity—detects as few as 50 cells
- Robust performance—highly referenced for cytotoxicity and viability assays
Fast & Simple Assay
- No cell lysis—ideal for use with time course experiments or post-measurement functional assays
- Flexibility—can be used with primary cells or cell lines, with adherent cells and cell suspensions
- Scalable—mix-and-read, homogeneous assay enhances speed while minimizing effort
- Compatible with fluorescence- or absorbance-based instrumentation
Economical and Safe to Use
- Economical—requires less reagent to get results comparable to the competition
- Safe—Nontoxic, nonradioactive reagent that is safe for the user, the cells, and the environment
alamarBlue® is Quantitative
The alamarBlue® assay is quantitative with respect to time and dose (Figure 1) as seen by the ability of metabolically active cells to convert the reagent into a fluorescent and colorimetric indicator.
图1. Cytotoxicity dose response using alamarBlue®. Growth inhibition data were used to determine the LD50for a toxic compound under investigation. Increasing concentrations of the compound proportionally corresponding decrease in alamarBlue® signal as cell metabolic activity was reduced.
alamarBlue® Response is Linear & Sensitive
The fluorescence-based readout makes alamarBlue® highly sensitive and as few as 50 cells in a 96-well plate can be detected with relatively short incubations.
To evaluate the sensitivity of alamarBlue®, a serial dilution of HUVEC cells was performed in a black, clear-bottom 96-well plate. Cells were then treated with alamarBlue® and the fluorescence measured 40 minutes and 18 hours later. After 40 minutes the fluorescence intensity of alamarBlue® is directly proportional to cell number in the range of 500–50,000 cells (Figure 2A). Further incubation of the same 96-well plate with reagent (18 h) leads to more sensitive detection in the range of 50–5,000 cells per well (Figure 2B) without significant increases in background fluorescence.
图 5a. Linearity of alamarBlue®. The fluorescence readout using alamarBlue® was linear over the range from ~500 to 50,000 cells after a 40 minute incubation.
图 5b. Sensitivity of alamarBlue®. This graph of the same 96-well plate shows that the limit of detection of alamarBlue® can reach 50 cells per well with linearity to 5,000 cells per well by using an 18 h incubation. The horizontal line at ~450 RFU represents the background fluorescence, calculated as three times the standard deviation of the “no cell” control.
alamarBlue®—Proven Performance in Viability and Cytotoxicity Assays
alamarBlue® has a proven track record as an indicator of cell health and its nontoxic nature permits long-term exposure of cells without negative impact; cells grown in the presence of alamarBlue® were found to produce similar numbers of viable cells as control cells, as determined by flow cytometric analysis of CD44, CD45RB, and CD4 antigens. These features make alamarBlue® an ideal assay for viability and cytotoxicity assays. See below for references where alamarBlue® has been used.
alamarBlue® Does Not Require Cell Lysis
alamarBlue® easily enters into live cells, eliminating the need to lyse or further process cells using fixation and DNA denaturation techniques. The dye is stable in cell culture media, including media containing phenol red. These characteristics allow you to:
- Continuously monitor the effects of drugs in a dose response assay or when optimizing drug concentrations (Figure 3)
- Perform other functional assays after cell viability measurements with alamarBlue® are complete
 | 图3. Toxicity analysis on HepG2 cells and SH-SY5Y cells. Cells were plated in 384-well plates and treated with varying concentrations of tamoxifen at total volume of 40 µL. After 24 hours 4 µL of alamarBlue® was added to each well. The signal was then read on a fluorescence microplate reader after one hour at 37ºC. Fluorescence intensity values were normalized to show difference in tamoxifen sensitivity. Data was fit to a sigmoidal dose response curve using GraphPad Prism. |
alamarBlue® Works Well With Different Types of Cells
Unlike some other reagents, alamarBlue® works well with either eukaryotic or prokaryotic cells and adherent or suspension cells. In addition, alamarBlue® is highly effective with a wide-range of primary cells. We have tested many primary cell types, including including HASmC, HUVEC, and HPAEC primary cells, and several of Invitrogen’s Cascade Biologics™ primary cell lines. We have also tested transformed cell lines, like SH-SY5Y cells. See figures through out this page for the data.
Primary cells and human hepatocytes from Invitrogen
Click for a complete list of primary cell lines available from Life Technologies. For more information on human hepatocytes and liver fractionations useful for toxicity testing go to CellzDirect, now part of the Life Technologies family.
alamarBlue® is Easy-To-Use and Scalable
The homogeneous assay format is based on a mix and read protocol, eliminating washing, fixation, and extraction steps; the single reagent is added directly to either cell suspension or attached cells in full medium. The protocol is readily amenable to automation and so particularly useful for high-throughput assays. The ability to detect alamarBlue® by either fluorescence or absorbance spectroscopy provides compatibility with a range of instrument platforms from microscopes, spectrometers and low and high-throughput plate readers.
See a protocol summary demonstrating how alamarBlue® can easily be used with HTS platforms.
alamarBlue® is Economical
alamarBlue® is an economical choice if you are currently using CellTiter® products. Results are correlative with CellTiter® Glo with similar sensitivity and identical to those obtained with CellTiter® Blue. However, you need to use only 1/10th as much alamarBlue as CellTiter® Glo, and ½ as much alamarBlue as CellTiter® Blue. While packaging appears to be similar, CellTiter® Blue is packaged as 5X compared to alamarBlue® packaged as a 10X solution. This means that you need to add twice as much CellTiter® Blue as you would alamarBlue® to get the same results. Switching to alamarBlue® can result in substantial cost savings without sacrificing sensitivity or accuracy.
alamarBlue® is Safe
Unlike the tritiated thymidine incorporation or chromium release assay, there is no radioactivity handling hazard associated with alamarBlue®. This trusted reagent is both non-radioactive and non-carcinogenic, ensuring safety for users, simple disposal, and lowers the impact on the environment compared to radioactive and other carcinogenic reagents.
alamarBlue® is Quantitative
The alamarBlue® assay is quantitative with respect to time and dose (Figure 1) as seen by the ability of metabolically active cells to convert the reagent into a fluorescent and colorimetric indicator.
图1. Cytotoxicity dose response using alamarBlue®. Growth inhibition data were used to determine the LD50for a toxic compound under investigation. Increasing concentrations of the compound proportionally corresponding decrease in alamarBlue® signal as cell metabolic activity was reduced.
alamarBlue® Response is Linear & Sensitive
The fluorescence-based readout makes alamarBlue® highly sensitive and as few as 50 cells in a 96-well plate can be detected with relatively short incubations.
To evaluate the sensitivity of alamarBlue®, a serial dilution of HUVEC cells was performed in a black, clear-bottom 96-well plate. Cells were then treated with alamarBlue® and the fluorescence measured 40 minutes and 18 hours later. After 40 minutes the fluorescence intensity of alamarBlue® is directly proportional to cell number in the range of 500–50,000 cells (Figure 2A). Further incubation of the same 96-well plate with reagent (18 h) leads to more sensitive detection in the range of 50–5,000 cells per well (Figure 2B) without significant increases in background fluorescence.
图 5a. Linearity of alamarBlue®. The fluorescence readout using alamarBlue® was linear over the range from ~500 to 50,000 cells after a 40 minute incubation.
图 5b. Sensitivity of alamarBlue®. This graph of the same 96-well plate shows that the limit of detection of alamarBlue® can reach 50 cells per well with linearity to 5,000 cells per well by using an 18 h incubation. The horizontal line at ~450 RFU represents the background fluorescence, calculated as three times the standard deviation of the “no cell” control.
alamarBlue®—Proven Performance in Viability and Cytotoxicity Assays
alamarBlue® has a proven track record as an indicator of cell health and its nontoxic nature permits long-term exposure of cells without negative impact; cells grown in the presence of alamarBlue® were found to produce similar numbers of viable cells as control cells, as determined by flow cytometric analysis of CD44, CD45RB, and CD4 antigens. These features make alamarBlue® an ideal assay for viability and cytotoxicity assays. See below for references where alamarBlue® has been used.
alamarBlue® Does Not Require Cell Lysis
alamarBlue® easily enters into live cells, eliminating the need to lyse or further process cells using fixation and DNA denaturation techniques. The dye is stable in cell culture media, including media containing phenol red. These characteristics allow you to:
- Continuously monitor the effects of drugs in a dose response assay or when optimizing drug concentrations (Figure 3)
- Perform other functional assays after cell viability measurements with alamarBlue® are complete
| 图3. Toxicity analysis on HepG2 cells and SH-SY5Y cells. Cells were plated in 384-well plates and treated with varying concentrations of tamoxifen at total volume of 40 µL. After 24 hours 4 µL of alamarBlue® was added to each well. The signal was then read on a fluorescence microplate reader after one hour at 37ºC. Fluorescence intensity values were normalized to show difference in tamoxifen sensitivity. Data was fit to a sigmoidal dose response curve using GraphPad Prism. |
alamarBlue® Works Well With Different Types of Cells
Unlike some other reagents, alamarBlue® works well with either eukaryotic or prokaryotic cells and adherent or suspension cells. In addition, alamarBlue® is highly effective with a wide-range of primary cells. We have tested many primary cell types, including including HASmC, HUVEC, and HPAEC primary cells, and several of Invitrogen’s Cascade Biologics™ primary cell lines. We have also tested transformed cell lines, like SH-SY5Y cells. See figures through out this page for the data.
Primary cells and human hepatocytes from Invitrogen
Click for a complete list of primary cell lines available from Life Technologies. For more information on human hepatocytes and liver fractionations useful for toxicity testing go to CellzDirect, now part of the Life Technologies family.
alamarBlue® is Easy-To-Use and Scalable
The homogeneous assay format is based on a mix and read protocol, eliminating washing, fixation, and extraction steps; the single reagent is added directly to either cell suspension or attached cells in full medium. The protocol is readily amenable to automation and so particularly useful for high-throughput assays. The ability to detect alamarBlue® by either fluorescence or absorbance spectroscopy provides compatibility with a range of instrument platforms from microscopes, spectrometers and low and high-throughput plate readers.
See a protocol summary demonstrating how alamarBlue® can easily be used with HTS platforms.
alamarBlue® is Economical
alamarBlue® is an economical choice if you are currently using CellTiter® products. Results are correlative with CellTiter® Glo with similar sensitivity and identical to those obtained with CellTiter® Blue. However, you need to use only 1/10th as much alamarBlue as CellTiter® Glo, and ½ as much alamarBlue as CellTiter® Blue. While packaging appears to be similar, CellTiter® Blue is packaged as 5X compared to alamarBlue® packaged as a 10X solution. This means that you need to add twice as much CellTiter® Blue as you would alamarBlue® to get the same results. Switching to alamarBlue® can result in substantial cost savings without sacrificing sensitivity or accuracy.
alamarBlue® is Safe
Unlike the tritiated thymidine incorporation or chromium release assay, there is no radioactivity handling hazard associated with alamarBlue®. This trusted reagent is both non-radioactive and non-carcinogenic, ensuring safety for users, simple disposal, and lowers the impact on the environment compared to radioactive and other carcinogenic reagents.
Analysis of cell proliferation and cytotoxicity is a vital step in evaluating cellular health and in the drug discovery process. alamarBlue is a proven cell viability indicator that uses the natural reducing power of living cells to convert resazurin to the fluorescent molecule, resorufin. The active ingredient of alamarBlue (resazurin) is a nontoxic, cell permeable compound that is blue in color and virtually nonfluorescent. Upon entering cells, resazurin is reduced to resorufin, which produces very bright red fluorescence (Figure 4). Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability—and cytotoxicity.
Figure 4. alamarBlue mechanism as an indicator. Resazurin, a non-fluorescent indicator dye, is converted to bright red–fluorescent resorufin via the reduction reactions of metabolically active cells. The amount of fluorescence produced is proportional to the number of living cells.
Using alamarBlue is very easy. Simply add alamarBlue mix-and-read solution to your cells, incubate for 1-4 hours, and read the fluorescence or absorbance (Figure 5). 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 innate metabolic activity and thus generate a proportionally lower signal than healthy cells.
alamarBlue is compatible with multiple instrument platforms. After incubation with alamarBlue, your samples can readily be measured on fluorescence and absorbance instrumentation. For fluorescence, simply set up your plate reader or fluorescence spectrophotometer using 560EX nm/590EM nm filter settings. Alternatively, the absorbance of alamarBlue can be read on a UV-Vis spectrophotometer at 570 nm.
Finally, analyze results by plotting fluorescence intensity (or absorbance) versus compound concentration. While results are linear and quantitative for both fluorescence and absorbance, fluorescence readings provide higher sensitivity.
alamarBlue is compatible with multiple instrument platforms. After incubation with alamarBlue, your samples can readily be measured on fluorescence and absorbance instrumentation. For fluorescence, simply set up your plate reader or fluorescence spectrophotometer using 560EX nm/590EM nm filter settings. Alternatively, the absorbance of alamarBlue can be read on a UV-Vis spectrophotometer at 570 nm.
Finally, analyze results by plotting fluorescence intensity (or absorbance) versus compound concentration. While results are linear and quantitative for both fluorescence and absorbance, fluorescence readings provide higher sensitivity.
Comparison with CellTiter–Glo and CellTiter-Blue
In side-by-side experiments using manufacturer's recommended procedures, alamarBlue was compared to CellTiter-Glo or to CellTiter-Blue in both primary cells (HUVEC) and transformed cell lines (SH-SY5Y). Figure 6A and 6B show that in both cell types alamarBlue gave a response comparable (in sensitivity and IC50) to CellTiter-Glo but only 1/10th of alamarBlue reagent was needed.
Similarly, when alamarBlue was compared to CellTiter-Blue, alamarBlue gave similar results (in sensitivity and IC50) to CellTiter-Blue and requires only 1/2 of the amount of reagent (Figure 7).
Comparison with MTT
Previous studies have shown that alamarBlue gives improved sensitivity and performance when compared to MTT assays. To read more about these findings, please check out this paper "Comparison of alamar blue and MTT assays for high through-put screening." by Hamid R, Rotshteyn Y, Rabadi L, Parikh R, Bullock P.
Figure 6A. alamarBlue v. CellTiter –Glo. alamarBlue (closed triangles) or CellTiter –Glo (open triangles). HUVEC cells were treated with tamoxifen for 24 hours prior to performing the cytotoxicity assays.
Figure 6B. alamarBlue v. CellTiter–Glo. alamarBlue (closed triangles) or CellTiter –Glo (open triangles) in SH-SY5Y cells.
Figure 7. alamarBlue v. CellTiter-Blue.
alamarBlue References
| Antczak, C. et al. (2007) High-throughput identification of inhibitors of human mitochondrial peptide deformylase. J Biomol Screening 12:521–535. |
| Al-Nasiry, S. et al. (2007) The use of alamarBlue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Hum Reprod 22:1304–1309. |
| Gartlon, J. et al. (2006) Evaluation of a proposed in vitro test strategy using neuronal and non-neuronal cell systems for detecting neurotoxicity. Toxicol In Vitro 20:1569–1581 |
| Ovcharenko, D. et al. (2005) High-throughput RNAi screening in vitro: From cell lines to primary cells. RNA 11:985–993 |
| Hamid, R. et al. (2004) Comparison of alamarBlue and MTT assays for high through-put screening. Toxicol In Vitro 18:703–10. |
| Gloeckner, H. et al. (2001) Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye alamarBlue. J Immunol Meth 252:131–138. |
| Nociari, M.M. et al. (1998) A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J Immunol Meth 213:157–167. |
| Nakayama, G.R. et al. (1997) Assessment of the alamarBlue assay for cellular growth and viability in vitro. J Immunol 204:205–8. |