WST-8 assay for high throughput screening of cell viability: comparison studies with MTT assay for monitoring cell growth

Many cell culture experiments involve large number of samples to be analyzed, either for monitoring cell growth or evaluation of the toxicity of compounds to the cells. There is an increasing interest to find rapid and accurate cell counting methods to the successful cell and tissue culture studies. The objective of this research was to compare accuracy, reliability, and reproducibility of a common cell counting method, MIT assay to the relatively new method, WST-8 assay for monitoring cell growth. Standard curve method was made by loading serially diluted CHO-Kl cells into a 96 well plate. The cell number was correlated with absorbance signals produced by MIT and WST-8 assays. With initial density of 1 x 103 to 4 x 103 cells/well, the cell growth of CHO-Kl cells was monitored either using trypan blue exclusion method or MTT and WST-8 assay for certain period of time (n=3). Results showed that WST-8 assay had a better correlation with cell number compared to MTT assay for monitoring cell growth. Using acid isopropanol as solubilisation agent, MTT assay gave a linearity of 0.9578 in the range of 400 to 1 x 105 cells/well with a maximum standard error of 11 %. DMSO did not increase the sensitivity and reliability of the method (r2 value was 0.9819 within a range of cell concentration of 1500 to 1 x 105 cells/well and has a standard error of 14 %). The WST-8 assay gave a better linearity with r2 value of 0.995 in the range of 400 to 8 x 104 cells/well with standard error less than 11%. During the cell growth, WST-8 assay is superior compared to MTT assay. It was concluded that its low sensitivity, high variability and technical consideration made MTT assay inappropriate to be regarded as the best method for cell counting. The soluble and ready to use tetrozolium salt, WST-8 assay offered higher accuracy and linearity for determining viable cell, making this assay more reliable and convenient for high throughput cell counting. Keywords: WST-8 assay âMTT assay âcell number and viabilit

Antioxidant effects of insulin-like growth factor-I (IGF-I) in rats with advanced liver cirrhosis

BACKGROUND: The exogenous administration of Insulin-like Growth Factor-I (IGF-I) induces hepatoprotective and antifibrogenic actions in experimental liver cirrhosis. To better understand the possible pathways behind the beneficial effect of IGF-I, the aim of this work was to investigate severe parameters involved in oxidative damage in hepatic tissue from cirrhotic animals treated with IGF-I (2 μg. 100 g(-1). day(-1)). Iron and copper play an important role in oxidative mechanisms, producing the deleterious hydroxyl radical (*OH) that peroxides lipid membranes and damages DNA. Myeloperoxidase (MPO) and nitric oxide (NO) are known sources of free radicals and induce reduction of ferritin-Fe(3+ )into free Fe(2+), contributing to oxidative damage. METHODS: Liver cirrhosis was induced by CCl(4 )inhalation in Wistar male rats for 30 weeks. Healthy controls were studied in parallel (n = 10). Fe and Cu were assessed by atomic absoption spectrometry and iron content was also evaluated by Perls' staining. MPO was measured by ELISA and transferrin and ferritin by immunoturbidimetry. iNOS expression was studied by immuno-histochemistry. RESULTS: Liver cirrhosis was histologically proven and ascites was observed in all cirrhotic rats. Compared to controls untreated cirrhotic rats showed increased hepatic levels of iron, ferritin, transferrin (p < 0.01), copper, MPO and iNOS expression (p < 0.01). However, IGF-treatment induced a significant reduction of all these parameters (p < 0.05). CONCLUSION: the hepatoprotective and antifibrogenic effects of IGF-I in cirrhosis are associated with a diminution of the hepatic contents of several factors all of them involved in oxidative damage

Chimeric Vitronectin : Insulin-like Growth Factor Proteins Enhance Cell Growth and Migration through Co-Activation of Receptors

Complexes comprised of IGF-I, IGF-binding proteins and the ECM protein vitronectin (VN) stimulate cell migration and growth and can replace the requirement for serum for the ex vivo expansion of cells, as well as promote wound healing in vivo. Moreover, the activity of the complexes is dependent on co-activation of the IGF-I receptor and VN-binding integrins. In view of this we sought to develop chimeric proteins able to recapitulate the action of the multiprotein complex within a single molecular species. We report here the production of two recombinant chimeric proteins, incorporating domains of VN linked to IGF-I, which mimic the functions of the complex. Further, the activity of the chimeric proteins is dependent on co-activation of the IGF-I- and VN-binding cell surface receptors. Clearly the use of chimeras that mimic the activity of growth factor:ECM complexes, such as these, offer manufacturing advantages that ultimately will facilitate translation to cost-effective therapies