alamarBlue - Tips and Advice

Sizes Available

alamarBlue can be used for applications such as functional assay, immunofluorescence, and ELISA.

This reagent is offered in two sizes:

25 ml = Enough for 2,500 wells
/96-well plate

100 ml = Enough for 10,000 wells
/96-well plate


Note: Calculations assume 100 µl final volume per well (96-well plate).

Overview

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.

Optimizing your experiments using these tips will improve your assay accuracy to achieve better results using alamarBlue:

  • Growth curves and repeated measurements
  • Which media to use?
  • The effects of phenol red
  • The effects of microbial and protein contaminants
  • Optimum incubation time
  • Storage conditions
  • Plate storage effects on absorbance readings

Growth Curves and Repeated Measurements

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.


Which Media to Use?

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.

Powdered Media Sigma Product Code Wavelength (nm)
               540             570 600 630
OX RED

OX     

RED    OX     RED   OX     RED 
BME EBSS B9638 0.61 1.207 0.853 1.502 0.845 0.244 0.261 0.177
BME HBS B9763 0.468 1.087 0.705 1.403 0.817 0.154 0.254 0.097
McCoy's 5A M4892 0.52 1.133 0.74 1.421 0.756 0.25 0.236 0.183
MEM EBSS M0268 0.582 1.186 0.819 1.483 0.82 0.235 0.252 0.168
MEM HBSS M4642 0.48 1.066 0.713 1.383 0.811 0.145 0.251 0.088
Nut Mix F-10 N6635 0.361 0.784 0.583 1.117 0.798 0.138 0.248 0.091
Nut Mix F-12 N6760 0.374 0.796 0.604 1.135 0.822 0.137 0.255 0.085
RPMI 1640 R6504 0.431 0.928 0.659 1.25 0.795 0.161 0.248 0.101

Table 3b. Fluorescence values for oxidized/reduced forms of alamarBlue for commonly used culture media.  

Powdered Media Sigma Product Code Fluorescence Units
                        Oxidized                            Reduced               
BME EBSS B9638 1,926 55,676
BME HBS B9763 3,840 60,256
McCoy's 5A M4892 2,640 50,545
MEM EBSS M0268 2,377 54,493
MEM HBSS M4642 4,194 59,202
Nut Mix F-10 N6635 2,472 70,092
Nut Mix F-12 N6760 5,232 68,132
RPMI 1640 R6504 6472 58,796

Note: 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.


The Effects of Phenol Red

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. 

Media   

Media

Absorbance

Value

Absorbance  Value for alamarBlue at Various Levels of Reduction                                               

 

  0% 10% 30% 60% 90% 100%
RPMI 1640 without
phenol red
0.032 0.47 0.52 0.61 0.73 0.85 0.88
RPMI 1640 with
phenol red
0.061 0.53 0.54 0.64 0.76 0.88 0.91

Note: 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 100 µl per well.


Microbial and Protein Contaminants

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) affect 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).


Optimum Incubation Time

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 104 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).


Storage Conditions

Store at +4oC. DO NOT FREEZE. This product should be stored undiluted and is photosensitive and should be protected from light. Please see label for expiry date.


Plate Effects on Absorbance Readings

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. 

Plate Type Day Absorbance
Blue (Oxidized)        Red (Reduced) 
540 nm 570 nm

600 nm

630 nm   540 nm  570 nm  600 nm  630 nm
Dynatech Flat Bottom Day 1

(0.003)

0.298

(0.005)

0.496

(0.007)

0.708

(0.003)

0.236

(0.02)

0.693

(0.027)

1.017

(0.002)

0.126

(0.00)

0.075

  Day 2

(0.003)

0.294

(0.004)

0.484

(0.006)

0.692

(0.002)

0.227

(0.02)

0.697

(0.027)

1.018

(0.008)

0.164

(0.009)

0.149

  Day 3

(0.003)

0.296

(0.006)

0.486

(0.008)

0.691

(0.003)

0.231

(0.010)

0.734

(0.024)

1.038

(0.008)

0.199

(0.009)

0.149

Corning Flat Bottom Day 1

(0.002)

0.21

(0.004)

0.335

(0.005)

0.474

(0.002)

0.169

(0.02)

0.53

(0.024)

0.772

(0.003)

0.137

(0.004)

0.105

  Day 2

(0.002)

0.21

(0.003)

0.329

(0.004)

0.458

(0.001)

0.161

(0.02)

0.58

(0.027)

0.822

(0.004)

0.193

(0.004)

0.159

  Day 3

(0.002)

0.2

(0.003)

0.322

(0.005)

0.444

(0.002)

0.16

(0.02)

0.6

(0.035)

0.823

(0.004)

0.21

(0.003)

0.172

Corning Round Bottom Day 1

(0.001)

0.38

(0.002)

0.635

(0.002)

0.913

(0.001)

0.3

(0.014)

0.87

(0.018)

1.266

(0.002)

0.151

(0.00)

0.084

  Day 2

(0.002)

0.39

(0.003)

0.641

(0.004)

0.914

(0.002)

0.295

(0.011)

0.85

(0.014)

1.241

(0.002)

0.146

(0.002)

0.083

  Day 3

(0.004)

0.39

(0.006)

0.646

(0.008)

0.916

(0.004)

0.302

(0.017)

0.86

(0.021)

1.237

(0.007)

0.159

(0.006)

0.094

Note: 100 µl of RPMI 1640 with MOPS pH 7.0, no phenol red. Standard deviations are in parentheses (calculated for n=8). 




References