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. 2002 May 20;195(10):1303–1316. doi: 10.1084/jem.20011624

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Copyright © 2002, The Rockefeller University Press

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Figure 9. — FasL expression on MVs purified from melanoma cell supernatant. (A) Western blot analysis. Analysis of MVs isolated from melanoma cell supernatant by serial ultracentrifugations, as performed by Western blot and staining with G247 mAb. An ∼35-kD band can be observed in MVs from melanoma lines (lanes 4, 6, and 7), resembling the one observed in MVs from PHA-activated Jurkat cells (lane 2). Only MV from PHA-activated, and not from resting Jurkat cells (lane 3) showed positivity for FasL, suggesting that this molecule unlikely derived from FBS used for cell culture. (B) Purity controls of microvesicle preparations. Controls performed to check purity of microvesicle preparations from melanoma cell supernatant revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, ER, or mitochondria. (C) Cytofluorimetric analysis of FasL expression on purified MVs. MVs from 501mel line were stained with anti-FasL NOK-1, anti-gp100, anti–LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. MVs were detected as granules with a size range of 100–600 nm, relative to standard beads of 6 μm size.

Figure 9. — FasL expression on MVs purified from melanoma cell supernatant. (A) Western blot analysis. Analysis of MVs isolated from melanoma cell supernatant by serial ultracentrifugations, as performed by Western blot and staining with G247 mAb. An ∼35-kD band can be observed in MVs from melanoma lines (lanes 4, 6, and 7), resembling the one observed in MVs from PHA-activated Jurkat cells (lane 2). Only MV from PHA-activated, and not from resting Jurkat cells (lane 3) showed positivity for FasL, suggesting that this molecule unlikely derived from FBS used for cell culture. (B) Purity controls of microvesicle preparations. Controls performed to check purity of microvesicle preparations from melanoma cell supernatant revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, ER, or mitochondria. (C) Cytofluorimetric analysis of FasL expression on purified MVs. MVs from 501mel line were stained with anti-FasL NOK-1, anti-gp100, anti–LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. MVs were detected as granules with a size range of 100–600 nm, relative to standard beads of 6 μm size.