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Assess Viability

Some treatments can affect viability in GLUTag cells, inducing abnormally high levels of GLP-1 secretion that can lead to misinterpretation of the results. Both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the neutral red uptake assays have been routinely performed in our laboratory to test the effects of new treatments on cell viability. Both are colorimetric assays and a reduction in absorbance corresponds with loss of cell viability. These tests are not quantitative and the addition as a reference of a positive control, such as hydrogen peroxide, is required.

Experimental Readout of the System

In secretion assays, GLP-1 content in medium and cells should be determined independently. Both medium and cell lysate should be collected and acidified to ensure preservation of peptides and facilitate the subsequent purification step. Our laboratory routinely extracts peptides from the medium and cell lysate by reversed-phase adsorption to C18 silica columns after acidification with trifluoroacetic acid. GLP-1 levels can then be determined by radioimmunoassay, ELISA or alternative quantification methods. GLP-1 secretion must be calculated as the total GLP-1 content of the medium divided by the total GLP-1 content of medium plus cells. A ratio of secretion is then obtained, which can be normalized to the percentage of secretion of the control group. A twoto fourfold increment in secretion can be found for the most potent secretagogues, such as the combination of forskolin and 3-isobutyl-1methylxanthine, which are often included in the study as positive controls.

In expression assays, medium is discarded and cells are scraped in a solution containing lysis buffer and β-mercaptoethanol to inactivate RNAses. Our laboratory has found that extraction of RNA using RNeasy Plus Mini Kit with QIAshredder (Qiagen, MD) permits collection of high quality RNA with a 260/280 nm ratio over 2 and 30–100 μg of RNA per 10 cm plate. In real-time PCR analysis involving cDNA derived from GLUTag cells, H3f3a amplicon (H3 histone gene) is strongly recommended as an internal control, since it shows much better consistency than other primers commonly used as housekeeping genes, such as 18S amplicons (personal observations). For protein extraction, medium is discarded and cells are scraped in a lysis buffer such as RIPA (radioimmunoprecipitation assay) and then sonicated. As much as 100 μg of protein per well can be collected from a 12-well plate.

 
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