EZ BlueTM Cell Assay Kit, EZ BlueTM solution is a resazurin based solution equivalent to Alamar Blue(R)

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EZ Blue™ Cell Assay Kit

Product Code: HM-CCK004

Product Description:

Cell proliferation and death are essential processes for tissue generation and regeneration, organ development etc. in mammals and are usually under stringent control of extra and intracellular factors. Non-physiological alterations in levels of these factors lead to anomalous cytogenetic behavior of cells which in turn leads to cell transformation, uncontrolled cell growth - the initiating event for cancer development. Pharmaceutical research is hence largely focused on effects of drugs, cytotoxic agents and biologically active compounds which affect cytogenetics.

Multiple procedures are available for determination of cell proliferation and cytotoxicity. Simple and cheap methods for estimating cell viability (or death) are Trypan Blue exclusion and Erythrocin B staining. However, these methods are not sensitive enough and cannot be used for high throughput screening. Measuring the uptake of radioactive substances, usually tritium-labeled thymidine, is accurate but it is also time-consuming and involves handling of radioactive substances. Quantitative measurement of reduction of Resazurin dyes is an alternate simple, safe and accurate method for determination of cell proliferation. This assay is similar to tetrazolium assay, except that it has the optional advantages of using fluorescent detection methods and omission the of solubilization step.

The EZBlue™ Cell Assay Kit is based on reduction of the water soluble, redox dye EZBlue™. The dye itself is dark blue in color and has intrinsic fluorescence. The fluorescence changes when EZBlue™ is reduced by metabolically active cells to the pink resorufin product. The spectral properties of resorufin allows the molecule to be detected using either absorbance or fluorescence. However fluorescence is the preferred method of evaluating the end-point of the reaction as it is more sensitive than absorbance. Dead or non-viable cells are incapable of this reduction and do not contribute to the fluorescence generated by viable cells. The specific mechanism responsible for the reduction of EZBlue™ is unknown, but probably involves the same cellular processes which generate equivalents such as NADH. In comparison to other methods of evaluating cell viability, which require tedious steps like reagent transfer, washing, crystal dissolution, repeated aspiration etc., the use of EZBlueTM is a single step process of addition of the reagent to cells. This ensures a rapid assay with minimal errors and facilitates its use in large scale hi-throughput screenings.


Applications:
 

• Cell proliferation: Quantification of changes in proliferative activity of cells caused by trophic factors, cytokines, radiations and growth promoters
• Cell cytotoxicity: Evaluation of effects of inhibitors or inducers of apoptosis, cytotoxic reagents, carcinogens and toxins
• Drug discovery: High-throughput screening of various anti-cancer drugs

• Cells in appropriate medium
• Adjustable pipettes and a repeat pipettor
• 96-well plate for culturing the cells
• 96-well plate reader or spectrophotometer capable of measuring the absorbance at 580nm and >600nm or fluorescence at 560nmEX/590nmEM

• Assay controls
  • Include appropriate assay controls i.e.
    1. Medium control (medium without cells)
    2. Cell control (medium with cells but without the experimental drug/ compound)
    3. Vehicle control (medium containing the experimental drug or compound but no cells)
• Accuracy
  • To obtain statistically significant data, perform the assay in triplicates or more.
  • Accuracy of the assay depends on pipetting skills of the personnel. Inappropriate addition and mixing practices may result in erroneous and false-positive or false-negative results.
• Incubation period
  • Different cell lines may respond in different manner to EZBlue™ depending on the properties of the cell lines, such as metabolic activity and doubling time. For this reason plating density and incubation period for every cell line should be optimized to obtain results in a linear range.
  • Although EZBlue™ does not affect the cell viability; incubation of cells with EZBlue™ for extended period may lead to reduction of fluorescent resorufin to colorless hydro-resorufin. Therefore determining the exact incubation period is essential.
• Sterility
  • For slow growing cell lines, longer incubation times are recommended. In such case, care should be taken to maintain sterile environment throughout the incubation period. Contamination leads to incorrect results as microbial contaminants also have capacity to reduce EZBlue™ reagent. This may lead to misinterpretation of the results and failure of the entire assay.
• Culture medium
  • pH of the medium used for the assay should be between 7.0 to 7.4. Presence of 10% fetal bovine serum has no effect on the results when reduction is measured spectrophotometrically. However, it causes quenching of the fluorescence.
• Temperature
  • Temperature affects the performance of the assay because of its effect on enzymatic rates. It is crucial to run the assay at a uniform temperature to ensure reproducibility across a single plate or among stacks of several plates. Since absorbance or fluorescence reading are measured at room temperature, it is important to ensure adequate equilibration of assay plates after removal from the 37°C incubator to avoid differential temperature gradients. Stacking large numbers of assay plates in close proximity should be avoided to ensure complete temperature equilibration.

• Preparation of reagent
  Bring the reagent to room temperature before use.
• Preparation of cells
  Always use freshly harvested cells for assay. Seed the cells in a cell culture flask or dish in an amount appropriate for the assay and incubate at 37°C in a 5% CO₂ environment. Allow the cells to grow for up to 24 hours or till confluence is reached. Harvest the cells and use for assay. (Note: The quantity of the cell suspension to be seeded in the medium depends upon doubling time of individual cell lines and seeding density to be used in the assay.)
• Procedure for determination of optimum cell density and incubation period to be used in the assay
  Use the procedure given below to determine optimum plating density and incubation period for cell line. This is a preliminary experiment that should be performed before staring the actual assay.
  1. Harvest the cells as explained in the Preparation of Cells section.
  2. Seed 100µl cells in 96-well plate at various cell densities, above and below the cell density expected to be used for the assay.
  3. Add 10µl EZBlue™ reagent. (10µl equals to 10% of the total culture volume per well).
  4. Wrap the plate in aluminium foil and incubate at 37°C in a 5% CO₂ environment.
  5. Read the absorbance at 580nm keeping >600nm as a reference wavelength, or fluorescence at 560nmEX/590nmEM at each hour following plating for first 6 - 8 hours.
  6. Process the data and calculate percentage reduction of EZBlue™ by using formulae mentioned in the Processing of the Data section below. This data will yield two types of information.
     1. Determination of optimum cell density
        For each incubation time point, plot the percentage reduction versus seeding cell density. Refer to the following graph.

 

2. Determination of optimum incubation period
   For each cell density, plot the percentage reduction versus time period of incubation. Refer to the following graph.

 

• Assay Procedures
  • Procedure for determining cell proliferation:
    1. Seed 100µl cell suspension in a 96-well plate at the required density. Treat them with cell growth modifying agent. (Note: a. If the cell growth modifying agent is a cytokine, metabolite, growth factor or any other compound, add its required quantity in the culture system. b. If the cell growth modifying agent is any kind of radiation or waves, treat the cells with them for required period of time.)
    2. Incubate the plate at 37°C in a 5% CO₂ atmosphere for required period of time.
    3. After the incubation period, remove the plates from incubator and add EZBlue™ reagent to a final concentration of 10% of total volume. This volume should be same as the volume used while determining optimum cell density. Wrap the plate with aluminium foil to avoid exposure to light.
    4. Return the plates to the incubator and incubate for 1 to 8 hours. (Note: Incubation time varies for different cell lines. Incubation time should be kept constant while making comparisons.)
    5. Remove plate from the incubator after incubation, followed by mixing on a gyratory shaker.
    6. Read the absorbance at 580nm keeping >600nm as a reference wavelength, or fluorescence at 560nmEX/590nmEM.
    7. The plate can be reread at a later time in culture period as long as cells remain viable. (Note: This time depends on the properties of the cell line as well as nature of the compound under test.)
  • Procedure for determining cell cytotoxicity
    1. For adherent cells:
       Seed 100µl cell suspension in a 96-well plate at required cell density, without the test agent. Allow the cells to adhere to the culture plate for about 24 hours. Add the desirable quantity of the test agent
       For suspension cells:
       Seed 100µl cell suspension in a 96-well plate at required cell density. Add the test agent immediately in the desired quantity
    2. Incubate the plate for desired period at 37°C in a 5% CO₂ atmosphere.
    3. After the incubation period, remove the plates from incubator and add 10µl EZBlue™ reagent. (10µl equals to 10% of the total culture volume per well.)
    4. Wrap the plate with aluminium foil to avoid exposure to light.
    5. Return the plates to the incubator and incubate for 1 to 8 hours. (Note: Incubation time varies for different cell lines. Incubation time should be kept constant while making comparisons.)
    6. Remove plate from the incubator after incubation, followed by mixing on a gyratory shaker.
    7. Read the absorbance at 570nm keeping >600nm as a reference wavelength, or fluorescence at 560nmEX/590nmEM.
    8. The plate can be reread at a later time in culture period as long as cells remain viable. (Note: This time depends on the properties of the cell line as well as nature of the compound under test).

• EZBlue™ reagent is light sensitive. Store the reagent in amber colored bottle.
• If stored at 2-8°C, the reagent remains stable for about 12 months. Stability of the reagent can be extended for several more months by aliquoting and storing at -20°C.
• Use before expiry date given on the label.

• Measuring proliferation or cytotoxicity using EZBlue™ by spectrophotometry
  • Calculation of percentage difference in reduction between treated and control cells: Equation for calculation of percentage difference in reduction between treated and control cells:

• Calculation of percentage reduction of EZBlue™:
  Equation for calculation of percentage reduction of EZBlue™:

• Measuring proliferation or cytotoxicity using EZBlue™ by fluorescence
  • Calculation of percentage difference in reduction between treated and control cells:
    Equation for calculation of percentage difference in reduction between treated and control cells:

• Calculation of percentage reduction of EZBlue™:
  Equation for calculation of percentage reduction of EZBlue™:

• Measuring reduction in EZBlue™ with different spectrophotometric wavelengths
  • Measure the absorbance of medium control (i.e. medium without cells, with EZBlue™) at higher and lower wavelength.
    Similarly, measure the absorbance of only medium (i.e. medium without cells and without EZBlue™) at higher and lower wavelength.
    Subtract the absorbance values of medium control from absorbance value of only medium.

• Calculation of Correction Factor (CF):

• Calculation for percentage reduction of EZBlue™ with different wavelength:

• Procedure
  1. CHO / Jurkat T cells (0.4 X 106 ) cells were seeded in triplicate wells of a 96-well plate.
  2. Medium without cells was used as blank control.
  3. EZBlueTM reagent and competitor reagent was added to the cells as per instructions provided.
  4. Cells were incubated for varying time intervals and absorbance was measured at 580 and 630 nm respectively and values for percentage reduction were calculated using the formulae provided in section 9(C).
  5. Percentage reduction values were then converted to number of cells using linear regression analysis or using the formula: Cell number = (Initial cell number x percentage reduction) + Initial cell number.
  6. The cell number determined from the experiment was then plotted against the time of incubation.
  7. Exponential growth curves were fitted using GraphPad Prism software.
• Observations

• Analysis

Cell numbers as calculated from values of percentage reduction:

Reagent	Initial cell density (million cells/ml)	Cell number at different time intervals
		0 Hour	2 Hours	3 Hours	4 Hours	24 Hours
EZBlue™	0.40	0.44	1.897	2.577	3.534	8.711
Competitor	0.40	0.543	2.039	2.591	3.589	11.078

• Results
  Doubling times were determined after curve fitting.

• Time saving: Ready to use EZBlueTM solution saves time of working by eliminating the steps like reagent transfer, washing, crystal dissolution and repeated aspiration
• Compatibility with multiple instruments: Reduced EZBlueTM can be detected by spectrophotometer or fluorescence detector depending on availability.
• Real-time monitoring of cell viability: Non-toxic nature of EZBlueTM allows real-time monitoring of cell viability
• Flexibility: EZBlueTM works on adherent as well as suspension cell lines
• Cell recovery: In contrast to MTT assay, EZBlueTM does not lead to cell death. Consequently, cells can be recovered and used in further analysis after incubation with EZBlueTM

• EZcountTM MTT Cell Assay Kit