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. Author manuscript; available in PMC: 2010 Feb 1.

Published in final edited form as: Traffic. 2008 Oct 31;10(2):218–234. doi: 10.1111/j.1600-0854.2008.00853.x

(a). Naive, wt and A53T αS over expressing MES cells conditioned in standard serum-containing medium and labeled with 2.5 μM FM1-43 fluorescent dye (molecular probes Eugene, OR) for 4 minutes. Access of dye was removed, and 10 minutes later the cells were fixed and tested for intracellular FM1-43 fluorescence. Pictures were taken in confocal, laser scanning microscope (Zeiss LSM 410) under non-saturating conditions. (b). Naïve, wt and A53T αS over expressing MES cells were conditioned for 16–18 hours in serum-free medium supplemented with 50μM BSA, followed by labeling with FM1-43 as in (a). (c). Cells as in (b) but conditioned in serum free medium supplemented with BSA only (50 μM) plus 250 μM 18:3 PUFA. (d). Quantitative presentation of the intracellular FM1-43 in BSA vs. PUFA treated cultures. Quantitation was performed on the captured images at selected image plane with the greatest intracellular FM1-43 signal, using ImageJ software, measuring the average fluorescence intensity (above threshold) across the entire cell divided by the area of the tested cell. Mean of 10–15 cells ± SD. * significant over the corresponding treatment in naïve cells and ** significant over the corresponding treatment in wt αS over expressing cells. TTEST P value < 0.05 (e) Cells were treated as in (b) and (c) but with 20 μM BSA with or without 100 μM 18:3 respectively. (f) Western blot of cytosolic extract from naïve, wt and A53T over expressing cells, treated for oligomers detection (49) and probed with anti αS H3C ab. Bar represents 10 μm.

(a). Naive, wt and A53T αS over expressing MES cells conditioned in standard serum-containing medium and labeled with 2.5 μM FM1-43 fluorescent dye (molecular probes Eugene, OR) for 4 minutes. Access of dye was removed, and 10 minutes later the cells were fixed and tested for intracellular FM1-43 fluorescence. Pictures were taken in confocal, laser scanning microscope (Zeiss LSM 410) under non-saturating conditions. (b). Naïve, wt and A53T αS over expressing MES cells were conditioned for 16–18 hours in serum-free medium supplemented with 50μM BSA, followed by labeling with FM1-43 as in (a). (c). Cells as in (b) but conditioned in serum free medium supplemented with BSA only (50 μM) plus 250 μM 18:3 PUFA. (d). Quantitative presentation of the intracellular FM1-43 in BSA vs. PUFA treated cultures. Quantitation was performed on the captured images at selected image plane with the greatest intracellular FM1-43 signal, using ImageJ software, measuring the average fluorescence intensity (above threshold) across the entire cell divided by the area of the tested cell. Mean of 10–15 cells ± SD. * significant over the corresponding treatment in naïve cells and ** significant over the corresponding treatment in wt αS over expressing cells. TTEST P value < 0.05 (e) Cells were treated as in (b) and (c) but with 20 μM BSA with or without 100 μM 18:3 respectively. (f) Western blot of cytosolic extract from naïve, wt and A53T over expressing cells, treated for oligomers detection (49) and probed with anti αS H3C ab. Bar represents 10 μm.