alamarBlue® general tips and advice

alamarBlue® general tips and advice

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®.

  • 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.

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 3a: Absorbance values for oxidized/reduced forms of alamarBlue® for commonly used culture media at different wavelengths.

Powdered Media Sigma Product Code Fluorescence Units
                        Oxidized                            Reduced               
BME EBSS B9638 1926 55676
BME HBS B9763 3840 60256
McCoy's 5A M4892 2640 50545
MEM EBSS M0268 2377 54493
MEM HBSS M4642 4194 59202
Nut Mix F-10 N6635 2472 70092
Nut Mix F-12 N6760 5232 68132
RPMI 1640 R6504 6472 58796

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.


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).

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

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.


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) 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).


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 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®).


Storage conditions

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.


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. 

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

600nm

630nm   540nm  570nm  600nm  630nm
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

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). 


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alamarBlue® is manufactured for Bio-Rad by Trek Diagnostic System. U.S. patent 5,501,959.