Measuring Cell Viability

The “EZ4U assay cell viability assay” was highlighted in a recent study investigating the effects of RNA modifications (m⁶A demethylases) on renal cell carcinoma cell lines. Accumulating evidence shows that m⁶A RNA methylation participates in the pathogenesis of multiple diseases including cancers. 

Renal cell carcinoma (RCC) accounts for approximately 3% of all cancers in adults. The 5-year survival rate for patients with locally advanced or metastatic RCC is poor with a prognosis of approximately 5%. Despite advanced pharmacological treatments of these patients (e.g. checkpoint immunotherapy, anti-VEGF antibodies, tyrosine kinase inhibitors), patient survival rates have only moderately improved.

Epigenetics studies the modification of heritable gene expression without having changes in the DNA sequence (1). N⁶-methyladenosine (m6A) is a common post-transcriptional methylation occurring in different types of RNAs in eukaryotic cells (2). m6A methylation is important in a number of physiological processes including the response to DNA damage. Recent studies suggest that RNA methylation has a great impact in cancer development and may provide a novel target for cancer treatment (3).

In a recent study researchers investigated the effects of RNA modifications (m⁶A demethylases) on renal cell carcinoma cell lines (4). Cell viability was determined with the BIOMEDICA EZ4U (easy for you) assay. In brief, cells were incubated with the EZ4U substrate for 3 hours at 37°C and absorbance was measured at 450 nm with 620 nm as reference.

Measuring Cell Viability with the EZ4U (MTT) Assay (developed and manufactured by Biomedica)

The EZ4U (easy for you) assay is a metabolic assay (similar to the MTT assay) that can quantify cell health. It measures the  reduction of the colorimetric substrate through the activity of mitochondrial enzymes in the cells.

Assays measuring cell viability are a widely used tool in cell biology research. The EZ4U test kit is a straightforward and reliable non-radioactive assay that is completed within two to five hours, depending on the cell type studied. The EZ4U assay employs non-toxic tetrazolium salts, which are, during incubation, converted into colored formazan. As the reduction process relies on functional mitochondria that become inactive shortly after cell death, this method effectively distinguishes between living and dead cells. The assay procedure is identical to the thymidine incorporation procedure. Therefore, no changes in test protocols are necessary. Furthermore, an additional benefit is the ability to continue cultivation after determining cell numbers.

The EZ4U cell proliferation and cytotoxicity assay is highly suited for testing cell viability in a number of different cell types. The assay has been used in various cell types and citations can be found in the link.

EZ4U Cell Proliferation and  Cytotoxicity Assay –single incubation step using living cells

Brief description: 20µl of the EZ4U substance (colorless non-toxic tetrazolium salts) is first added to each well. The mitochondria of the living cells oxidize the EZ4U substance to a yellow-colored formazan derivate. The color change is measured on a conventional plate reader at 450 nm using a reference wavelength of 620 nm.

EZ4U  Cell Viability & Cytotoxicity Assay (cat.no. BI-5000)

• Reliable & Sensitive
• One step incubation  – for use on living cells
• Widely used – referenced in +270 publications

 

Download the BROCHURE – EZ4U cell proliferation and cytotoxicity assay and the PROTOCOL BOOKLET

Literature

1. The role of m6A modification in the biological functions and diseases. Jiang X et al., Signal Transduct Target Ther. 2021; 21;6(1):74. PMID: 33611339.
2. Depletion of the m⁶A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma. Hu W et al., Am J Transl Res. 2023; 15(3):1744-1755. PMID: 37056835.
3. The potential role of RNA N6-methyladenosine in Cancer progression. Wang T et al., Mol Cancer. 2020. 19(1):88. PMID: 32398132.
4. Depletion of the m⁶A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma. Hu W Am J Transl Res. 2023 Mar 15;15(3):1744-1755. PMID: 37056835.