99 research outputs found
GREEN FLUORESCENT PROTEIN (GFP), SUATU SIGNAL PENANDA QUANTITATIF UNTUK MEMONITOR PROLIFERASI SEL
Get PDFBanyak penelitian sel kultur yang melibatkan sejumlah besar sampel untuk dianalisis, baik yang berhubungan dengan pertumbuhan sel atau toksisitas senyawa terhadap sel. Green Fluorescent Protein (GFP) adalah suatu protein yang secara alami dapat berfluorescence dan banyak digunakan pada berbagai aplikasi seperti penanda untuk ekspresi gena, produksi heterologous protein atau monitoring efisiensi transfeksi. Penelitian ini bertujuan untuk membandingkan tingkat akurasi, taraf kepercayaan dan reprodusibilitas GFP untuk memonitor pertumbuhan sel.Kurva baku dibuat dengan serial dilusi sel CHO-K1-EGFP dalam media 10% FCS di 96 well plate. Jumlah sel dalam tiap sumuran dihubungkan dengan signal fluorescence. Untuk memonitor pertumbuhan sel, signal fluoresensi dibandingkan dengan metode Trypan Blue Exclusion yang jumlah sel dalam tiap sumuran dihitung selama periode waktu tertentu (n=3). Untuk monitoring pertumbuhan sel, signal dari GFP memperlihatkan korelasi yang baik dengan jumlah sel dengan tingkat linieritas 0,9866 dalam kisaran jumlah sel 1250 – 1 x 105 sell/sumuran (standar error maksimum 11%). Metode ini terbukti memungkinkan pengukuran langsung signal fluoresensi sehingga mampu menurunkan kemungkinan kesalahan yang terjadi pada saat preparasi sel yang dapat mempengaruhi akurasi data yang diperoleh. Sekali klones permanen (stable clones) diperoleh klons ini dapat digunakan untuk banyak aplikasi.Kata Kunci: Green Fluorescent Protein (GFP), Fluorescence, proliferasi sel, Trypan Blue Exclusio
WST-8 assay for high throughput screening of cell viability: comparison studies with MTT assay for monitoring cell growth
Get PDFMany 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
Thermoelectric Response Near the Density Driven Mott Transition
Full text linkWe investigate the thermoelectric response of correlated electron systems
near the density driven Mott transition using the dynamical mean field theory.Comment: 4 pages, 2 embedded figure
Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence
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