GFP-MVI was expressed in a variant of PC12 cells (ATCC, Cat. No CRL-1721.1) as described above. These variant cells are not responding to stimulating factors (such as KCl or NGF) but are better attached to the surface. FRAP was carried out using Leica TCS SP5 laser confocal microscope with HCX PL APO 63×/1.25-0.75 Oil Cs objective. GFP fluorescence was excited using the 488 nm line of the Ar ion laser and emission was collected between 500 and 560 nm at a 1 Airy disc pinhole size. Pixel size was 122 nm × 122 nm. Scanning was performed at 400 – 800 Hz with laser power set to 35% (gain 720V; offset 0.5-3%). Bleaching was carried out with laser power set to 100%. FRAP experiments were performed and analyzed using the FRAP-wizard tool of the Leica Confocal Software Version 2.6.1 Build 1537 (Leica Microsystems). In brief, recordings started by taking 5 frames at maximal speed (2.93 s/frame). Then a 7.44 nm × 7.44 nm region of interest (ROI) was bleached three times (using the “zoom in” option), and 10 frames were recorded at maximal speed followed by taking 5 frames at 5-s interval. For calculating the half-time of recovery, the resulting curves were fitted to a single exponential function (using FRAP-wizard tool). Signals in the ROI were background corrected. Half times of recovery were obtained from three independent experiments. Images were exported into Adobe Photoshop.
Frap wizard tool
Manufactured by Leica
The FRAP-wizard tool is a specialized instrument designed for performing Fluorescence Recovery After Photobleaching (FRAP) experiments. The core function of this tool is to accurately measure the diffusion and mobility of fluorescently labeled molecules within a defined region of interest in a sample. The FRAP-wizard tool provides researchers with the necessary capabilities to conduct these types of fluorescence-based studies.
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2 protocols using frap wizard tool
1
FRAP Analysis of GFP-MVI in Variant PC12 Cells
2
Macrophage Infection and Laser-Induced Amastigote Extrusion
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Macrophages obtained from C57BL/6-GFP mice or transfected RAW 264.7 macrophages were infected with L. amazonensis-DsRed2 amastigotes for 24 to 96 h and cultivated in round dishes, suitable for the incubator coupled to the Leica SP5 II TS system. Using the Leica FRAP Wizard tool provided by the acquisition software, we delimited an area or a region of interest (ROI), in which laser pulses were applied. These ROI corresponded to Hoechst-stained macrophage nuclear regions, adjusting the focal plane (z axis) to the centre of the nuclei; a concentrated near-ultraviolet (UV) laser [405 nm, at 100% power (50 mW) and 400 Hz frequency] was applied to the ROI for 300–500 s before multidimensional live recordings (Soustelle et al., 2008 ). The technique was also employed to hasten the extrusion of amastigotes from RAW transfected cells, which are not suitable for long-term in vitro infection (i.e. for more than 5 days).
To check whether micro-irradiation is inducing apoptosis, micro-irradiation of 48 h infected BMDMØ cultures was performed in complete medium supplemented with 5% of L929 cell conditioned medium, 2.5 mM CaCl2, 500 nM of nuclear probe YO-PRO-1 Iodide (Molecular Probes) and 2.5 μg ml−1 of Annexin V-CF633 (Biotium).
To check whether micro-irradiation is inducing apoptosis, micro-irradiation of 48 h infected BMDMØ cultures was performed in complete medium supplemented with 5% of L929 cell conditioned medium, 2.5 mM CaCl2, 500 nM of nuclear probe YO-PRO-1 Iodide (Molecular Probes) and 2.5 μg ml−1 of Annexin V-CF633 (Biotium).
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