ABSTRACT
Objective: To analyze multimodal magnetic nanoparticles-Rhodamine B in culture media for cell labeling, and to establish a study of multimodal magnetic nanoparticles-Rhodamine B detection at labeled cells evaluating they viability at concentrations of 10mug Fe/mL and 100mug Fe/mL. Methods: We performed the analysis of stability of multimodal magnetic nanoparticles-Rhodamine B in different culture media; the mesenchymal stem cells labeling with multimodal magnetic nanoparticles-Rhodamine B; the intracellular detection of multimodal magnetic nanoparticles-Rhodamine B in mesenchymal stem cells, and assessment of the viability of labeled cells by kinetic proliferation. Results: The stability analysis showed that multimodal magnetic nanoparticles-Rhodamine B had good stability in cultured Dulbecco's Modified Eagle's-Low Glucose medium and RPMI 1640 medium. The mesenchymal stem cell with multimodal magnetic nanoparticles-Rhodamine B described location of intracellular nanoparticles, which were shown as blue granules co-localized in fluorescent clusters, thus characterizing magnetic and fluorescent properties of multimodal magnetic nanoparticles-Rhodamine B. Conclusion: The stability of multimodal magnetic nanoparticles-Rhodamine B found in cultured Dulbecco's Modified Eagle's-Low Glucose medium and RPMI 1640 medium assured intracellular mesenchymal stem cells labeling. This cell labeling did not affect viability of labeled mesenchymal stem cells since they continued to proliferate for five days.
Objetivo: Analisar a estabilidade das nanopartículas magnéticas multimodais-Rhodamine B em meios de cultura para marcação celular e, consequentemente, estabelecer o estudo de detecção intracelular de nanopartículas magnéticas multimodais-Rhodamine B nas células marcadas, avaliando a viabilidade celular nas concentrações de 10mig Fe/mL e 100mig Fe/mL. Métodos: Foram realizados: análise da estabilidade das nanopartículas magnéticas multimodais-Rhodamine B em meios de cultura diferentes; marcação das células-tronco mesenquimais com nanopartículas magnéticas multimodais-Rhodamine B; detecção intracelular das nanopartículas magnéticas multimodais-Rhodamine B nas células-tronco mesenquimais e avaliação da viabilidade das células marcadas por meio da cinética de proliferação. Resultados: A análise de estabilidade determinou que as nanopartículas magnéticas multimodais-Rhodamine B presentes nos meios de cultura Dulbecco's Modified Eagle's-Low Glucose e RPMI Medium 1640 apresentaram boa estabilidade. A marcação das células-tronco mesenquimais com nanopartículas magnéticas multimodais-Rhodamine B descreveu localização intracelular das nanopartículas, as quais se mostraram como grânulos azulados colocalizados nos grumos fluorescentes, caracterizando, assim, as propriedades magnéticas e fluorescentes das nanopartículas magnéticas multimodais-Rhodamine B. Conclusão: A estabilidade das nanopartículas magnéticas multimodais-Rhodamine B, presentes nos meios de cultura Dulbecco's Modified Eagle's-Low Glucose e RPMI Medium 1640, garantiu a eficiente marcação intracelular das células-tronco mesenquimais. Esse tipo de marcação não afetou viabilidade das células-tronco mesenquimais marcadas, já que as mesmas continuaram proliferando ao longo de 5 dias.
Subject(s)
Mesenchymal Stem Cells , Nanoparticles , RhodaminesABSTRACT
Objective To evaluate the normalized correction effect of the ROX reference fluorescence on the real-time quantitative RT-PCR assessment for the Nrf2 mRNA.Methods The Taq-man probe was utilized in the real-time quantitative RT-PCR method to assess the Nrf2 mRNA; The reaction specificity of the Nrf2 PCR products obtained from the rat cDNA was assessed using gel electrophoresis and sequencing identification.The PCR calibrators with different concentrations varied at 10 gradients(from 2.0 × 109 to 2.0 × 100) were prepared for real-time quantitative RT-PCR assessment,and then the influence of the ROX reference fluorescence correction on the linear range of the PCR standard curve was investigated experimentally.The recombinant plasmids with different concentration levels(e.g,high,medium or low concentration) were assessed by the real-time RT-PCR and 20 replicate measurements were performed for all the samples in both intra- and inter-assays.The repeatability differences of both the inter- and intra-assays were analyzed systemically with/without the ROX reference fluorescence correction.Results The primers and probes required for Nrf2 mRNA real-time quantitative RT-PCR assessments were constructed,and the reaction system and conditions were also established.The length of the amplified fragment agreed well with the expected length(122 bp),and the sequencing analysis showed the amplified fragment was verified by the sequencing identification.After applying the ROX reference fluorescence correction,the linear range ofthe PCR standard curve was measured as 2.0 × 109 -2.0 × 100 copies/μl(R2 ＞ 0.99),which was 100times wider than that measured without ROX reference fluorescence correction(2.0 × 109 - 2.0 × 102copies/μl,R2 ＞ 0.99).Without the ROX reference fluorescence correction,the intra-assay CVs of the Ct value were measured as 4.1%,2.7% and 2.1% for the recombinant plasmids with high-,medium-,and low-level concentration,respectively.The inter-assay CVs of the Ct value were 4.3% 、3.0% 、2.4%.By contrast,with the ROX reference fluorescence correction,the intra-assay CVs of the Ct value became 0.7%,0.5%,0.4% and the inter-assay CVs of the Ct value turned to 1.0%,0.8% and 0.7%.The discrete degree of the Ct value with the correction was lower than that without the correction.The intra- and interassay Ct values of the high- and medium-level samples exhibited statistic significance for the measurements with/without the correction.In the high-level group,the t values of the intra- and inter-assays were 4.843 and 2.566,with P＜0.05.In the medium-level group,the t values of the intra- and inter-assays were 4.293 and 4.423,with P＜0.05.However,no statistic significance was observed the low-level group(the t values of the intra- and inter-assays were 0.753 and 1.279,with P ＞.0.05).Conclusions The utilization of the ROX reference fluorescence in the real-time quantitative PCR assessment for Nrf2 mRNA could widen the linear range of the standard PCR calibration curve and improve the repeatability of the assessment,which should be quite helpful for accurately assessing the expression level of the Nrf2 mRNA.