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Fluorescence Recovery After Photobleaching: live cells
Author: Steven Boeynaems, updated date: , view: 24, Q&A: 0
Tags: Microscopy, phase separation and membraneless organelles

Materials and Reagents

  1. Cell line of choice (e.g., HeLa or U2OS cells work well for most purposes because of contact inhibition and large cytoplasm)

  2. DMEM + 10% FBS + pen/strep (or medium of choice)

  3. Lipofectamine 3000

  4. Plasmids containing fluorescent fusion proteins of choice (or stable cell line carrying fluorescent transgene)

  5. Ibidi glass bottom dishes (μ-dish, high, #81158)


Equipment

  1. LSM 780 Meta NLO confocal microscope (ZEISS, model: LSM 780 NLO ), with temperature and CO2 control and incubation chamber.

  2. Standard cell culture inubator

Procedure

  1. Seed cells at a 70% confluency in Ibidi dishes, and incubate overnight at 37°C and 5% CO2 in incubator.

  2. Transfect 3μg of plasmid with lipofectamine 3000 according to manufacturer's instructions, and incubate overnight at 37°C and 5% CO2 in incubator (in case of using stable cell lines: omit and proceed to step 3).

  3. Start microscope half an hour before imaging to let incubation chamber equilibrate to 37°C and 5% CO2.

  4. Transfer Ibidi dish to sample holder on 40x oil objective and fix. Use small drop of immersion oil. Set focus and wait for 5min to let system equilibrate. This will reduce chance that field of focus will start drifting during imaging.

  5. Look for cells with membraneless organelles or aggregates through the ocular.

  6. Focus on a cell and take a snap shot with the Zen software.

  7. Highlight three circular regions of interest (ROI). Diameter depends on whether full-droplet or intr-droplet bleaches are pursued. ROI 1 = area to be bleached. ROI 2 = background outside cell. ROI 3 = bleach control of similar intensity in field of view. Record fluorescence intensity for these three areas, but only bleach ROI 1.

  8. Settings: Choose smallest time intervals between measurement. Bleach after three baseline measurements at 100% power until 50% drop in intensity is reached.

  9. Start experiment and measure for at least 30 sec but preferably 1 min. This depends on how mobile the organelles are.

  10. Export measurements as a .txt file.








Data analysis and summary

  1. Import data from text file to excel sheet.

  2. Subtract the fluorescence intensity of the background from the bleached droplet and reference droplet.

  3. Subtract the fluorescence intensity of the first time point post-bleach from the bleached droplet. This step will normalize the first post-bleach time point to zero.

  4. Divide the fluorescence intensities of the bleached droplet by the pre-bleach fluorescence intensity. Do this as well for the reference droplet. This will normalize the pre-bleach time point to 1.

  5. Divide the fluorescence intensities of the bleached droplet by the fluorescence intensities of the reference droplet. This will correct for photobleaching due to prolonged exposure during the time course experiment. 

  6. Import the normalized data into GraphPad Prism.

  7. Plot the fluorescence intensities as a function of time.

  8. Average FRAP curves of at least 15 droplets and evaluate differences between multiple conditions using two-way ANOVA.


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