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FACS analysis on cellular uptake of synthetic S41 peptide
Recently, it has been postulated that apparent CPP internalization can be caused by fixation artifacts [26]. To exclude this for S41, we obtained a FITC-labeled synthetic S41 peptide and examined its internalization ability in living N2A XL888 by flow cytometry. Low temperature causes the lipid membrane to become more rigid and minimizes active processes of cellular transport and should inhibit uptake of true CPPs. As shown on Fig. 3A and B, S41 uptake into cells was greatly inhibited when performed at 4 °C indicating that uptake of S41 is temperature-dependent and that internalization involves active processes such as endocytosis.
Fig. 3.
The influence of temperature, MβCD and heparinase III (Hep III) on the internalization of FITC labeled S41 into N2A cells. FITC-labeled S41 were incubated with N2A cells at different temperatures or with MβCD and Hep III, and then subjected to flow cytometry. A and C show fluorescence scans while B and D show the average fluorescence. Means ± SD are indicated. Each sample was analyzed in duplicate or triplicate. Values significantly (p
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