Developed & Manufactured by Biomedica
  • home
  • products
  • EZ4U – Cell Proliferation and Cytotoxicity Assay | BI-5000

EZ4U – Cell Proliferation and Cytotoxicity Assay | BI-5000

Cat. No.: BI-5000

1 kit / €  200,00Please Inquire

Contact distributor

EZ4U Cell Proliferation Assay and Cytotoxicity Test Highlights:
Therapeutic Areas
EZ4U Cell Proliferation and Cytotoxicity Assay methods comparison
EZ4U Cell Proliferation and Cytotoxicity Assay cell lines comparison
Assay Characteristics
  • Method

    Cell proliferation and cytotoxicity assay, 10 x 96 tests, method based on the reduction of tetrazolium salt to colored formazan

  • Sample type

    Cell culture

  • Sample volume

    200 µl / test

  • Assay time

    2 – 5 hours, depending on the metabolic capacity of the cells

  • Use

    Research use only

Product details

EZ4U – Cell Proliferation Assay Product Overview

The EZ4U kit is a two to five-hour test for the measurement of cell proliferation and cell toxicity. The EZ4U test employs non-toxic tetrazolium salts, which are reduced to colored formazan. As this reduction process requires functional mitochondria, which are inactivated within a few minutes after cell death, this method provides an excellent tool for the discrimination between living and dead cells.

EZ4U – Cell Proliferation Assay Principle

The assay set-up is performed in the same manner as the standard 3H-thymidine incorporation method. Instead of pulsing with tritiated nucleotide, 20 µl of dye solution is added to 200 µl sample. The incubation time is dependent on the metabolic capacity of the cells. Usually two to five hours of incubation at 37°C are sufficient to yield a significant increase in color intensity. As different cells vary in their ability to convert the yellow colored tetrazolium compound to its red formazan derivative, we recommend testing every new cell-line's metabolic capacity as described in the figure below.


EZ4U kit Assay principle

Different metabolic capacity of various cell lines. 3 x 103 cells/well were cultivated in 200 µl RPMI 1640. Following a cultivation period of 3 days, 25 µl of the dye substrate were added to each well. Optical density was recorded after 4 hours, showing significant differences in the metabolic capacity of the various cell lines.

After incubation, the plate is removed from the incubator and gently mixed by tipping the plate towards all four sides. To avoid increased standard deviations, the plate must be shaken before reading the optical density.

The absorbance is measured on a microplate-reader set at 450 nm or 492 nm with 620 nm as a reference wavelength. The reference absorbance at 620 nm (or any wavelength between 620-690 nm) is used to correct for nonspecific background values caused by cell debris, fingerprints, or other potential interferences. However, the reference may be omitted without significant changes in the accuracy of the assay.

Method Comparison


EZ4U Method Comparison

EZ4U – Cell Proliferation Assay Typical Absorption Spectrum

The figure below shows a typical absorption spectrum for the EZ4U assay.


 Typical Absorption Spectrum EZ4U kit

EZ4U – Cell Proliferation Assay Kit Components

Contents

Description

Quantity

SUB

Substrate, lyophilized

10 vials

ACT

Activator solution, ready to use

1 x 30 ml

 

Package insert + QC sheet

1 piece each

Storage instructions: All reagents of the EZ4U kit are stable at 4°C until the expiry date stated on the label of each reagent.

Instructions for use

Reagent Preparation

One vial SUB contains enough substrate for 96 tests, i.e. one 96-well plate with 200 µl of cell culture medium/well. If more substrate is needed, combine the dissolved substrates prior to pulsing.

1.

Dissolve the SUB (substrate) in 2.5 ml ACT (activator).

2.

Prewarm this solution to 37°C prior to addition. If necessary, warm up the substrate vial in your hand while mixing with activator.
This procedure yields a straw-coloured solution.

The mixed substrate is designed for immediate use only and should not be stored.

EZ4U – Cell Proliferation Assay Protocol

1.

All reagents and samples must be at room temperature (18-26°C) before use in the assay.

2.

Add 200 µl cell culture into the respective wells.

3.

Add 20 µl SUB (substrate) into each well, swirl gently.

4.

Incubate at 37°C for 2-5 hours dependent on the metabolic capacity of the cells.

5.

If no microplate reader with a shaking plate carrier is available, mix the plate on a vibrating platform or by tipping it by hand.

6.

Read absorbance at 450 nm or 492 nm, with 620 nm as reference.

7.

To improve the accuracy of the results, absorbance from a substrate blank in assay medium without cells should be subtracted from all other values. 

Important Considerations

The substrate is not sterile. If sterile conditions are demanded, the solubilised ready-to-use dye substrate must be sterile filtered. (A minor turbidity prior to filtration interferes neither with the filtration, nor with the assay performance).

Due to the high sensitivity of this test, it is advisable to use as few cells as possible. Otherwise a non-linear titration curve may result.

To achieve reproducible time kinetics in color development, equilibrate cell cultures at 37°C.

Do not prolong incubation times without prior testing, as this might result in an increased background without improved sensitivity.

The use of a reference wavelength of 620 nm (which is subtracted from the values obtained at 450 or 492 nm) is not strictly necessary but increases the performance of the test.

The chromophore appears to be non-toxic and therefore continuation of cell culture is possible after the removal of the formazan derivative.

Technical Hints

Do not mix or substitute reagents with those from other lots or sources.

Do not mix stoppers and caps from different reagents or use reagents between lots.

Do not use reagents beyond expiration date.

Protect reagents from direct sunlight.

Avoid foaming when mixing reagents.
Background & Therapeutic Areas

Proliferation assays are widely used in cell biology for the study of growth factors, cytokines, nutrients and for the screening of cytotoxic or chemotherapeutic agents. There are several ways to determine the number of cells either by microscopic inspection, through an electronic particle counter, indirectly by measuring the incorporation of radioactive precursors, quantifying total protein with chromogenic dyes, or by measuring metabolic activity of cellular enzymes. The most common assay for cell proliferation is the incorporation of 3H‑thymidine into cellular DNA. The 3H-thymidine assay is, however, labor-intensive as it requires the removal of the excess, unincorporated label by using some cell harvesting method before measurement. In 1956, the first paper on the use of tetrazolium salts as indicators of cell viability was published. The method was based on the finding that living cells are capable of reducing slightly colored or uncolored tetrazolium salts into intensely colored formazan derivatives. This reduction process requires functional mitochondria, which are inactivated within a few minutes of cell death. Therefore, this method provides an excellent tool for the discrimination of living and dead cells. However, the early tetrazolium salts did have some disadvantages, such as the insolubility of the resulting formazan products. Time and labor-consuming solubilization procedures were necessary, including repetitive pipetting and mixing, or the application of hazardous solubilizers. This necessary post-assay treatment, however, irreversibly terminated cell proliferation and thus made it impossible to prolong incubation in order to achieve an increase in sensitivity or continue cell culture. These inconve­niences led to the development of non-toxic tetrazolium salts which yield soluble reduction products. Although the assay procedure was made easier by these soluble dyes, in practice the use was limited due to the instability of the formazan dye and a relatively low absorbance of the end product as compared to the classical MTT assay.

The Biomedica research department has solved both problems and created an easy-to-use, rapid and reliable non-isotopic cell proliferation assay. For convenience, we have made it highly compatible with the standard thymidine incorporation assay. Therefore, no changes are required in the setup of the test and in the "labeling" procedure. Furthermore, there is no need for the removal of culture medium before or after the addition of the chromogenic substrate and neither solubilization nor harvesting procedures are necessary. The work performed by Biomedica resulted in an assay which combines the best of the thymidine and MTT methods, namely: accuracy, speed, reliability, and ease of use. Also, according to our data achieved so far, the chromophore appears to be non-toxic. A double labeling with EZ4U and a radioactive nucleotide to obtain more information about cell viability and DNA content is now feasible.

Citations

Powered by BizGenius
Reviews
1 Review
Sort by:
Submit a Review

  • Measuring Cell Proliferation with EZ4U

    27 July, 2021

    We wanted to check for the proliferation of murine T cells upon activation with anti CD3/CD28. This kit gave good result to indicate proliferation of activated T cells.

    Application: Proliferation assay

    Starting Material: Murine pan T cells

    Results Summary: The result showed proliferation of activated T cells

    The Good
    Easy and fast

    The Bad
    A bit difficult to reproduce the result

    The Bottom Line
    Easy to use kit

    Biocompare Product Review

    Lisha Joshi Medical University of Graz Molecular Biology and Biochemistry Research Scientist (Biocompare – Review) –

    EZ4U graph signal after incubation
Add a review

Your email address will not be published. Required fields are marked *


Downloads

Cat. No.: BI-5000

1 kit / €  200,00Please Inquire

Contact distributor

Request more information




Contact Us

Phone Head Office: +4312910711 Phone Product Support: +4312910745
img