Although alamarBlue® is a simple and easy to use reagent for cell proliferation and viability experiments, there are some key facts and tips which will help you get the best results in your research. Through optimizing your experiments using these alamarBlue® tips you can improve the assay accuracy and achieve better results using alamarBlue®.
alamarBlue® can be used for long term cell proliferation assays and for repeated measurements. For these types of study it is recommended that aliquots of the cell medium/ suspension are taken at each time point for incubation with alamarBlue® prior to an endpoint test. Please refer to Breinholt et al. (1998) for an example of this type of experiment.
96 well plates can be refrigerated and read within 1-3 days (Please refer to Appendix 4 in Technical data sheet), and re-heat plates to 37oC for fluorescence measurements.
Most commonly used media are suitable for use with alamarBlue® assays, please see table 3a and 3b below for specific examples.
Table 3a: Absorbance values for oxidized/reduced forms of alamarBlue® for commonly used culture media at different wavelengths.
Table 3b: Fluorescence values for oxidized/reduced forms of alamarBlue® for commonly used culture media.
Measurements were taken on a Cambridge Technology, Inc. (Watertown, MA) Model 7620 Microplate. Fluorometer-settings were: bottom reading, light source setting 12, no max AFU; Excitation: 530 nm; Emission: 580 nm, gain/16.
Studies have shown that there is no interference from the presence of phenol red in the growth medium. The presence of phenol red merely shifts the values approximately 0.03 units higher (please see Table 4).
Table 4: Effect of phenol red on absorbance values at 570 nm. Absorbance value for various levels of reduction of alamarBlue® in RPMI 1640 pH 7.0, with MOPS (both with and without phenol red) when using Dynatech flat bottom plates and 100µl per well.
alamarBlue® can be reduced by microbial contaminants. Therefore, results from contaminated cultures tested may not be accurate unless appropriate controls are in place.
In-house studies indicated samples with protein concentrations equivalent to 10% fetal bovine serum (FBS) did not interfere with the assay. However, the serum may cause some quenching of fluorescence and so serum controls should also be used Page et al. (1993). It has also been determined that increasing concentrations of FBS and bovine serum albumin (BSA) affects the assay. Therefore, a specific method and calculation was developed to analyze the test matrix for effects due to these two compounds; allowing the correction of such effects (Goegan et al. 1995).
alamarBlue® measures cell proliferation most accurately when the cells are in the exponential growth phase. Long incubation times can also lead to a decrease in the efficiency of alamarBlue® buffering agents. A cell density of 1 x 10e4 cells/ml is generally recommended, and a control experiment is advisable to determine the optimal cell density and incubation time for alamarBlue® (please see the working examples of how to use alamarBlue®).
This product is in liquid form and may be stored for 12 months at room temperature at 2-8oC for 20 months. This product should be stored undiluted; it is photosensitive and should be protected from light.
The absorbance readings of alamarBlue® may be affected by the type of plates which are used for experiments and so plate type should be a consideration when planning experiments involving alamarBlue®. A series of absorbance readings were taken for the oxidized and reduced forms of alamarBlue® using a range of different types of plates. Following the readings on day 1, plates were covered in foil and refrigerated prior to further readings on days 2 & 3.
Table 5. Absorbance of alamarBlue® oxidized and reduced forms for a range of plates, including those refrigerated prior to data collection. 100ul of RPMI 1640 with MOPS pH7.0, no phenol red. Standard deviations are in parentheses (calculated for n=8).
alamarBlue® is manufactured for Bio-Rad by Trek Diagnostic System. U.S. patent 5,501,959.
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