I strongly recommend doing a cell number titration and time of exposure to Alamar blue.  Some times 1 hr with the reagent is enough sometimes you need the full 4 hrs.  This depends on the metabolic activity of the cell line.  So each cell line will vary, and from lab-to-lab.  It is very important to have cell number and time so that the FL value is not too high so you do not see inhibition.  Use the linear part of the cell curve.

Start from 5,000 - 30,000 and do the titration AFTER growing for the the number of days you are planning to grow the cells.

Cell health can be monitored by numerous methods. Plasma membrane integrity, DNA synthesis, DNA content, enzyme activity, presence of ATP, and cellular reducing conditions are known indicators of cell viability and cell death. alamarBlue® cell viability reagent functions as a cell health indicator by using the reducing power of living cells to quantitatively measure the proliferation of various human and animal cell lines, bacteria, plant, and fungi allowing you to establish relative cytotoxicity of agents within various chemical classes. When cells are alive they maintain a reducing environment within the cytosol of the cell. Resazurin, the active ingredient of alamarBlue® reagent, is a non-toxic, cell permeable compound that is blue in color and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Viable cells continuously convert resazurin to resorufin, increasing the overall fluorescence and color of the media surrounding cells.

alamarBlue® cell viability reagent is used to assess cell viability by simply adding the 10X, ready-to-use solution to mammalian or bacterial cells in culture media. There is no requirement to aspirate media from cells or place cells in minimal media. Consequently, alamarBlue® reagent can easily be used in a single tube or microtiter plate format in a “nowash” fashion. Simply add alamarBlue® reagent as 10% of the sample volume (i.e., add 10 μL alamarBlue® reagent to 100 μL sample), followed by a 1–4 hours incubation at 37ºC. Longer incubation times may be used for greater sensitivity without compromising cell health (Figure 3 and see Frequently Asked Questions). The resulting fluorescence is read on a plate reader or fluorescence spectrophotometer. Alternatively, the absorbance of alamarBlue® reagent can be read on a spectrophotometer. Finally, results are analyzed by plotting fluorescence intensity (or absorbance) versus compound concentration.

alamarBlue® reagent

 alamarBlue® cell viability assay protocol. A 96-well plate containing the cells and the compounds to be tested is prepared using standard methods. alamarBlue® reagent is added directly to each well, the plates are incubated at 37°C to allow cells to convert resazurin to resorufin, and the fluorescence (or absorbance) signal is measured. Results are evaluated by plotting the fluorescent (or absorbance) signal versus compound concentration. This depicts an assay carried out in a 96-well plate, but the procedure is readily adaptable to other formats as well (including 384-well plates and tubes of various volumes). If tubes are used, the sample is transferred to a cuvette prior to spectrophotometric analysis.

alamarBlue® reagent can detect as few as 50 cells per well in a 96-well plate. To evaluate the sensitivity of alamarBlue® reagent, a serial dilution of HUVEC cells was performed in a black, clear-bottom 96-well plate. Cells were then treated with alamarBlue® reagent and the fluorescence was measured 40 minutes and 18 hours later. After 40 minutes, the fluorescence intensity of alamarBlue® reagent was directly proportional to cell number in the range of 500–50,000 cells. After 18 hours, the fluorescence intensity of alamarBlue® reagent was directly proportional to cell number in the range of 50–5,000 cells, leading to more sensitive detection 

Linearity of alamarBlue®. The fluorescence readout using alamarBlue® was linear over the range from ~500 to 50,000 cells after a 40 minute incubation.

Sensitivity of alamarBlue Figure 2B: 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.

Materials

Materials required but not provided

Mammalian or bacterial cells in appropriate medium

Appropriate 96- or 384-well plates

Optional: 3% SDS in phosphate buffered saline (PBS), pH 7.4

Preparing Cells

Mammalian Cells—Adherent: Plate mammalian cells in a cell culture flask or dish, and allow cells to adhere and grow for approximately 4–24 hours at 37°C and 5% CO2 before proceeding with the assay.

Mammalian Cells—Suspension: Plate mammalian cells in a cell culture flask or dish, and use cells immediately for the assay or allow cells to grow for up to 24 hours at 37°C and 5% CO2 before proceeding with the assay.

Bacterial Cells: Notes alamarBlue® reagent is stable to multiple freeze/thaw cycles and its activity is not affected if the reagent is frozen.

General Guidelines

Cell types assayed with alamarBlue® reagent include mammalian, bacterial2 (including biofilms3), plant,4 and fish cells.5 More specifically alamarBlue® reagent has been tested on hepatocytes, such as HepG2 cells, as well as cells of primary origin.5

Be sure to include appropriate assay controls. To minimize experimental errors, we recommend making measurements from a minimum of 4–8 replicates of experimental and no-cell control samples.

You may need to determine the plating density and incubation time for the alamarBlue® assay for each cell type and use conditions such that the assay is in the linear range.

If you plan to use longer incubation time (overnight), be sure to maintain sterile conditions during reagent addition and incubation to avoid microbial contaminants. Contaminated cultures will yield erroneous results as microbial contaminants also reduce alamarBlue® reagent.

Fetal bovine serum (FBS) and bovine serum albumin (BSA) cause some quenching of fluorescence. We recommend using the same serum concentration in controls to account for this quenching. Other media components, such as phenol red do not interfere with the assay.

Protocol

Optional: Treat cells with the test compound 24–72 hours prior to performing the alamarBlue® cytotoxicity assay.

Add 1/10th volume of alamarBlue® reagent directly to cells in culture medium 

Format

Volume of cells + medium

Volume of 10x alamarBlue to add

Cuvette

1 mL

100 µL

96-well plate

100 µL

10 µL

384-well plate

40 µL

4 µL

Incubate for 1 to 4 hours at 37°C in a cell culture incubator, protected from direct light.

Note: Sensitivity of detection increases with longer incubation times. For samples with fewer cells, use longer incubation times of up to 24 hours.

Record results using fluorescence or absorbance as follows:

Fluorescence: Read fluorescence using a fluorescence excitation wavelength of 540–570 nm (peak excitation is 570 nm). Read fluorescence emission at 580–610 nm (peak emission is 585 nm).

Absorbance: Monitor the absorbance of alamarBlue® at 570 nm, using 600 nm as a reference wavelength (normalized to the 600 nm value).

Note: Fluorescence mode measurements are more sensitive. When fluorescence instrumentation is unavailable, monitor the absorbance of alamarBlue® reagent. Assay plates or tubes can be wrapped in foil, stored at 4°C, and read within 1–3 days without affecting the fluorescence or absorbance values.

Optional: Add 50 μL 3% SDS directly to 100 μL of cells in alamarBlue® reagent to stop the reaction.

FAQ's

General Questions

Q: How does alamarBlue® work?

A: Healthy living cells maintain a reducing state within their cytosol. This “reducing potential” of cells converts alamarBlue® reagent into a detectable fluorescent (or absorbent) product.

Q: Is alamarBlue® reagent toxic?

A: No. alamarBlue® reagent is a safe, non-toxic reagent to both the sample and user.

Q: Does alamarBlue® reagent need reconstitution?

A: No, alamarBlue® reagent is supplied as a 10X, ready-to-use solution.

Q: Can I use alamarBlue® reagent with suspension cells too?

A: Yes. alamarBlue® reagent works on adherent and suspension mammalian cells.

Q: Can I use alamarBlue® reagent with non-mammalian cells, such as bacteria?

A: Yes, alamarBlue® reagent has been shown to work with bacterial2 and plant cells.4

Q: alamarBlue® reagent is not the most expensive cytotoxicity indicator on the market, does that mean it doesn’t work as well as other reagents?

A: Actually, alamarBlue® reagent is comparable to other often more expensive cytotoxicity indicators.

Q: Since alamarBlue® is an absorbance or fluorescence readout, is it as sensitive as a luminescence product?

A: alamarBlue® reagent is sensitive enough to detect less than 50 mammalian cells in a single well of a 96-well plate.

Storage Questions

Q: What if I left the alamarBlue® stock reagent at room temperature, overnight?

A: The reagent is stable for up to 12 months when stored at room temperature (~22ºC).

Q: I accidentally froze the alamarBlue® stock reagent, can I still use it?

A: Yes. alamarBlue® reagent is stable to multiple freeze/thaw cycles. Be sure to heat the reagent in a 37°C water bath and mix the reagent to ensure a homogenous solution before use.

Q: Do I need to protect alamarBlue® reagent from light?

A: Yes, alamarBlue® reagent is very slowly converted into a fluorescent product over time, when exposed to light, thus leading to high background values. Store the reagent, protected from light.

Methods Questions

Q: What is the optimal incubation time and temperature of cells with alamarBlue® reagent?

A: Incubate the cells with alamarBlue® reagent for 1–4 hours at 37°C. For more sensitive detection with low cell numbers, increase the incubation time for up to 24 hours.

Q: Can you incubate cells with alamarBlue® reagent overnight?

A: Yes. However, signals from higher cell density samples may have “saturated,” which means the linearity of reagent may have reached a plateau. If this occurs, decrease the incubation time.

Q: What if I don’t have an instrument suitable for reading fluorescence?

A: The absorbance of alamarBlue® reagent also changes depending on cell viability and proliferation. Therefore, simply monitor the absorbance of the reagent at 570 nm, while using 600 nm as a reference wavelength.

Q: Is alamarBlue® assay strictly an endpoint assay?

A: No. While alamarBlue® can be used as a terminal readout of a population of cells, the reagent can also be used to continuously monitor cell viability and proliferation in real time. Since alamarBlue® reagent is non-toxic, you can incubate cells with reagent and monitor fluorescence (or absorbance) over time on the same sample.

Troubleshooting Questions

Q: What is the problem for observing high background fluorescence values?

A: The reagent may be breaking down due to exposure to light. Be sure to store alamarBlue® reagent in the dark and do not expose the reagent to direct light for long periods of time.

Q: Why are the fluorescence values so low in intensity?

A: Try increasing the incubation time of cells with alamarBlue® reagent, changing the instrument’s “gain” setting, and checking the instrument filter/wavelength settings. Make sure to have positive controls (living cells) in the experimental design for troubleshooting.

Q: Why are the fluorescence values so high that they are beyond the linear range of the instrument?

A: Try decreasing the incubation time or reducing the number of cells used in the experiment.

References

1. Invest Ophthalmol Vis Sci 38, 1929 (1997)

2. Infect Immun 65, 3193 (1997)

3. J Antimicrob Chemother 57, 1100 (2006)

4. Phytochem Anal 12, 340 (2001)

5. Anal Biochem 344, 76 (2005)

Resazurin Assay

The Resazurin assay (also known as Alamar Blue assay) offers a simple, rapid and sensitive measurement for the viability of mammalian cells and bacteria. Living cells are metabolically active and are able to reduce the non-fluorescent dye resazurin to the strongly-fluorescent dye resorufin . The fluorescence output is proportional to the number of viable cells over a wide concentration range. This also allows the calculation of the proliferation rate for cells capable of consecutive cell division. Resazurin is effectively reduced in mitochondria making it also useful to assess mitochondrial metabolic activity.

www.promega.com/resources/protocols/technical-bulletins/101/celltiter-blue-cell-viability-assay-protocol/

I strongly recommend doing a cell number titration and time of exposure to Alamar blue.  Some times 1 hr with the reagent is enough sometimes you need the full 4 hrs.  This depends on the metabolic activity of the cell line.  So each cell line will vary, and from lab-to-lab.  It is very important to have cell number and time so that the FL value is not too high so you do not see inhibition.  Use the linear part of the cell curve.

Start from 5,000 - 30,000 and do the titration AFTER growing for the the number of days you are planning to grow the cells.

Try to seed 3000 or 5000 cells per well/100ul of medium. I think you are seeding too much cells and plates are getting confluent after 24 hours. Most of the cytotoxic drugs act on proliferating cells, so u need healthy and proliferating cells for this assay. Try not to incubate plates for more than 2 hours. Best of Luck.

Agree also with Sandeep.... I did not read your question fullY !!

50,000 cells is too much.  And 24 hrs with an inhibitor is too little time.  Go down to 1000 - 5000 cells for 3-4 days.  Then your differences will be more apparent.  The dye also requires good growing cells, and time of exposure with dye will need to be figured out.  Determine the linear portion of the curve first and then run the inhibition.

 You have to optimize the seeding density before proceeding with drug treatment. Signal to noise ratio, i.e. ratio of fluorescence of cells + media and media alone should be large enough to have a linear range for your compound. 5000-10000 cells may not work if your cells are slow growing.Or else increase the incubation time after addition of alamar blue. May be around 18 hours.

Hope this would help.