ABSTRACT
BACKGROUND: Fetal bovine serum (FBS) has been used to support the growth and proliferation of mammalian cells for decades. Owing to several risk factors associated with FBS, several trials have been conducted to evaluate substitutes to FBS with the same efficiency and the lower risk issues.METHODS: In this study, human platelet lysate (HPL) derived from activated human platelets was evaluated as an alternative to FBS due to the associated risk factors. To evaluate the efficiency of the preparation process, platelet count was performed before and after activation. The concentrations of several growth factors and proteins were measured to investigate HPL efficiency. HPL stability was studied at regular intervals, and optimal heparin concentration required to prevent gel formation in various media was determined. The biological activity of HPL and FBS was compared by evaluating the growth performance of Vero and Hep-2 cell lines.RESULTS: Result of platelet count assay revealed the efficiency of HPL preparation process. Growth factor concentrations in HPL were significantly higher than those in FBS, while the protein content of HPL was lower than that of FBS. Stability study data showed that the prepared HPL was stable for up to 15 months at −20℃. Ideal heparin concentration to be used in different media was dependent on calcium concentration. Results of cell viability assay showed that HPL was superior to FBS in supporting the growth and proliferation of Vero and Hep-2 cells.CONCLUSION: The HPL prepared by the mechanical activation of platelets may serve as an efficient alternative to FBS in cell culture process.
Subject(s)
Humans , Blood Platelets , Calcium , Cell Culture Techniques , Cell Line , Cell Survival , Heparin , Intercellular Signaling Peptides and Proteins , Platelet Count , Risk FactorsABSTRACT
Objective@#To develop a micro-neutralization test for determination of neutralizing antibody against ZIKA virus (ZIKV) in human sera and to verify the acute and convalescent serum samples of 10 ZIKA virus-infected cases diagnosed by nucleic acid detection and/or virus isolation.@*Methods@#ZIKV isolated from ZIKA cases was used to determine micro-neutralization antibody. The virus solution was prepared by infecting BHK21, VERO and VERO-E6 cell lines and viral titer was tested; 100 TCID50 viral solution and 4 times diluted sera which were inactivated at 56 ℃ for 30 min were neutralized, then added the cell suspension and incubated in 5% CO2 incubator at 37 ℃ for 7 d. The CPE was observed every day.@*Results@#The sensitivity of BHK21, VERO and VERO-E6 was different after infection with ZIKA virus. VERO cell line was the most sensitive and showed typical CPE. VERO cell line was used to establish a micro-neutralization test for determination of neutralizing antibody against ZIKA virus in sera.@*Conclusions@#The neutralizing antibody test for zika virus in sera is a special and usefulmethod to diagnose human infection of ZIKV and to conduct population based epidemiological investigation.
ABSTRACT
Renal disorders have become very common nowadays, which may also lead to kidney failure. The disorder may be caused by the commonly used chemicals such as acetaminophen, CCl4, streptromycin, H2O2 etc. The objective of this work is to determine the nephroprotective potential of ethanolic extract against H2O2 induced toxicity in VERO cell line. Ethanolic extract is known for its antioxidant, anti-inflammatory and anti-microbial effects, which make it a most sought for herbal medicine. Its characteristic features have identified this compound as a potential hepatoprotective and nephroprotective agent. VERO cells are cells taken from the kidney of an African green monkey, which are used in our study. The matured leaves of Melia Azadirachta were used to prepare ethanolic extract and the same was used to test for its inhibitory effect in 96 micro plate formats against in VERO cell lines. To study the cytotoxic properties of ethanolic extract against VERO cell line, we have tested the MTT assay with different concentrations in the range of 1000 to 62.5 μg/ml. From the performed assay, the effect of ethanolic extract drug reveals an enhanced activity on in VERO cell lines and that infers Melia Azadirachta, can be used as nephroprotective agent.
ABSTRACT
Objective To establish a stable transfection cell line of iRhom2 and its mutant through recombinant lentivirus infection.Methods The full-length gene of iRhom2 and its mutant were cloned into the lentivirus vector Lenti-OE-Flag, and got recombinant lentiviral vector of Lenti-OE-iRhom2 and Lenti-OE-iRhom2mut.The constructed recombi-nant lentivirus vectors were transfected into HEK-293T packaging cells to obtain the recombinant virus.Vero cells were in-fected with recombinant virus.The stable expressing cell lines were obtained by pressure screening with puromycin. Results The recombinant lentivirus vectors were constructed and the recombinant virus was obtained.The stable express-ing cell lines were obtained using virus infection and the protein expression was testified with Western blotting.Conclu-sions Stable iRhom2-expressing Vero cell line and its mutant are achieved by recombinant lentivirus infection.It paves the way for future study on biological functions and mechanism of iRhom2.