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alamarBlue Protocols

alamarBlue is a cell viability assay reagent which contains the cell permeable, non-toxic, and weakly fluorescent blue indicator dye resazurin. It has been extensively referenced and used in a wide range of research areas. alamarBlue quantitatively measures proliferation in human, animal, bacterial, fungal, and mycobacterial cells. It is useful for cytokine bioassays, cell viability assays, and in vitro cytotoxicity determinations as well as cell growth monitoring. Popular alamarBlue is a preferred alternative to other cell proliferation assays such as MTT, XTT, 3H Thymidine, or neutral red.

alamarBlue Work Principle

Resazurin is used as an oxidation-reduction (REDOX) indicator that undergoes colorimetric change in response to cellular metabolic reduction. The reduced form, resorufin, is pink and highly fluorescent, and the intensity of fluorescence produced is proportional to the number of living cells respiring. alamarBlue is a direct indicator of cell health and it detects the level of oxidation during respiration, quantitatively measuring cell viability and cytotoxicity.

alamarBlue Work Principle

Here are detailed protocols for use of alamarBlue.

Quality Control Testing Method for alamarBlue

AlamarBlue Cell Viability

Protocol for assaying cell viability with a test compound:

  1. When cells are in log phase of growth, harvest them and determine the cell count.
  2. Note: The suggested optimal cell count is 1 x 104 cells/ml. It may varybetween different cell lines.

  3. To appropriate wells of a 96 well plate, add the test compound and vehicle controls so that the final volume is 100µl in each well. Culture cells at 37°C in a cell culture incubator for desired test compound exposure period.
  4. Remove the assay plates from 37°C incubator and mix by gently shaking it.
  5. Aseptically add 10µl of alamarBlue Reagent in an amount equal to 10% of the volume in the well. In the Positive Control well, add 10µl of ultrapure sterile water. Incubate cultures at 37°C.
  6. Note: For positive control well, add 10µl of ultrapure sterile water.

  7. After the incubation time ends, remove the plate and measure the fluorescence with Excitation wavelength at 530-560nm and Emission wavelength at 590nm.

Determine LD50 Using alamarBlue

alamarBlue can be used to determine the median lethal dose LD50 value and similar assays testing the effects of a toxin, radiation, or pathogen on cell cultures. Use semi-log graph paper to plot the percent of untreated control for each dilution of a given test agent on the y-axis vs. the concentration of the test agent on the x-axis. To determine the LD50 endpoint from the graph, read from where the 50% intercepts the dose.

Response curve to the concentration along the x-axis. Fig.1 Response curve to the concentration along the x-axis.

Measuring Cytotoxicity or Proliferation Using alamarBlue

  1. When cells are in log phase of growth, harvest them and determine the cell count.
  2. Note: The suggested optimal cell count is 1 x 104 cells/ml. It may varybetween different cell lines.

  3. Plate cells and expose to test agent as determined by researcher. For determining the effect of a test agent on cell growth, ensure correct controls are included.
  4. Mix by shaking. Aseptically add alamarBlue in an amount equal to 10% of the volume in the well. Incubate cultures with alamarBlue for 4-8 h.
  5. Note: The optimum incubation time may vary between cell types.

  6. Measure cytotoxicity or proliferation using spectrophotometry or fluorescence.

Determining Optimum Length of Incubation and Plating Density

The two variables which most affect the response of cells to alamarBlue are length of incubation time and number of cells plated. It is suggested that the plating density and incubation time should be determined for each cell line using the following steps.

alamarBlue Spectrophotometry Calculations with Different Filters

  1. Make up alamarBlue as directed in the package insert.
  2. Measure the absorbance of alamarBlue in media at the lower wavelength (LW) filter and at the higher wavelength (HW) filter.
  3. Measure the absorbance of 100 μL media only (blank) at the two wavelengths.
  4. Subtract the absorbance values of media only from the absorbance values of alamarBlue in media.
  5. Note: AOLW = absorbance of alamarBlue in media - absorbance of media only, lower wavelength

    AOHW = absorbance of alamarBlue in media - absorbance of media only, higher wavelength

  6. Calculate correction factor (RO): RO=AOLW /AOHW
  7. Calculate percentage difference in reduction between treated and control cells in cytotoxicity/proliferation assays:
  8. Percentage difference in reduction= [ (A LW - (AHW x Ro) for test well ] x 100%/ A LW - ( AHW x Ro ) for control well.

    ALW = absorbance at lower wavelength minus the media blank

    AHW = absorbance at higher wavelength minus the media blank

    Ro = correction factor

  9. If the alternative calculation to find the percentage reduction of alamarBlue is required, use the following equation:
  10. Percentage reduction of alamarBlue = [A LW- ( AHW x Ro ) ] x 100%

Advantages of Using alamarBlue

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