The overall goal of the following experiment is to monitor chromatin protein dynamics in embryonic stem cells. This is achieved by transecting e ESL with GFP fusion plasma DNA to express fluorescently tagged chromatin protein in transfected cells, frap or flip is performed to measure the recovery or loss. After photobleaching, the protein's dynamic behavior is monitored and the mobile and immobile protein fractions are determined.
Results are obtained that show differences in chromatin protein dynamics based on frap and flip assays. The main advantage of this technique over existing methods, such as extraction of chromatin proteins using salsa detergents, is that it is applied for single living cells. Therefore, the association between chromatin and chromatin proteins is observed live, and the heterogeneity within a given population can be determined.
Begin by plating mouse, embryonic fibroblasts, or meth cells using sterile technique. In a culture hood, prepare the live imaging eight well micro slides with a code of gelatin. Allow the gelatin coating to fix for five to 30 minutes at room temperature and then aspirate away the free gelatin.
Now seed each coated well with 22, 000 meth cells in DMEM media supplemented with fetal fetal bovine serum. Allow cells to grow in a tissue culture incubator after six hours. Aspirate away the DMEM media now seed each meth coated well with 15, 000 R one embryonic stem cells in supplemented ES media.
Allow the cells to become 50%confluent by incubating overnight in the tissue culture incubator. The next day, the cells should have obtained confluence of 30 to 50%Start by replacing the old ES media with 250 microliters of fresh ES media. Next, prepare the transfection mixture to a 1.5 milliliter sterile test tube at 100 microliters of serum free media, followed by 10 microliters of transit.
LT one transection reagent. Mix the tube by gentle pipetting and incubated room temperature for five to 20 minutes. After five to 20 minutes, add 1.5 micrograms of GFP fusion plasma DNA, and mix by gentle pipetting.
Then incubate room temperature for another 15 to 30 minutes. With the transfection mixture prepared, add 13.5 microliters of it to each well and swirl the micro slides to ensure even dispersal. Allow the cells to incubate for another 24 to 48 hours in the incubator before continuing with the frap or flip analysis.
After 24 hours, replace the old ES media with 250 microliters of fresh ES media because many consecutive images are acquired. An and or revolution spinning disc confocal microscope with photobleaching capabilities is preferred. For wrap and flip experiments over any standard confocal microscope, choose the proper solid state laser according to the fluorescent protein to be visualized.
Adjust the objective to oil immersion. Put oil on the lens, place the cells, then close the lid of the chamber. Since live cells are going to be imaged, it is essential to use an environmental chamber to control oxygen, humidity, carbon dioxide, and temperature similar to tissue culture incubator conditions.
Observe the cells with blue fluorescence and select a cell expressing GFP. Using a 60 x oil immersion lens, ensure that the cell shows correct subcellular distribution. Occasionally when expression levels are too high, the proteins localization may spill to other compartments such as the nucleus.
Such cells should not be selected. Select a region of interest in the e chromatin or hetero chromatin compartment. The ladder is used as condensed GFP foci.
Now set up an imaging protocol. Set three to five frames before the photo, bleach the photo bleach, and 90 to 120 frames after the photo bleach with 250 to 1000 millisecond intervals, the laser intensity for the repeated frames should be at a minimum, which is usually around five to 10%if fluorescence is adequate. On the other hand, the laser intensity for the photobleach should be at maximum 80 to 100%Generally photobleach with a laser pulse of 20 to 40 microseconds.
All these numbers can change depending on the intensity of the laser, the analyzed protein and the expression levels. Now run the program when photobleaching is appropriate. You should observe a black hole in your GFP fluorescence.
The black hole is gradually refilled with fluorescence after recovery. For a flip experiment, set up a different imaging protocol. Collect three to five frames before bleaching, then repetitively photobleach and image at different intervals.
Depending on the GFP tagged protein used. Bleach and collect 120 images repeatedly throughout the entire experiment. For either technique.
Repeat the process on 20 to 30 cells per well and make three biological repeats, preferably on different days in homogenous populations and correct settings. The standard deviation across three experiments is usually around or below 5%in all wrap frames.Collected. Measure the fluorescence intensity in the region of interest, the non bleached to nuclear area, and the background area.
When the bleached region is negligible, the entire nucleus can be selected for normalization purposes. Collect these data as a function of time before and after bleaching. Now make some calculations.
Subtract the background from all data. Calculate ratios between the bleached and non bleached areas, and divide the bleached ratio by the pre bleached ratio. For preble images, you should get a value of approximately one.
The first image after the bleach will indicate the bleached depth. Subtract the value from one for the actual bleach depth value, repeat for every cell and average 20 to 30 cells from each experiment In all flip frames collected, measure the fluorescent intensity in the non bleach to nuclear area, a neighbor cell and the background area Calculation of flip data is similar to a FRA curve. Only the analyzed ROI should be different than the actual bleached region, which is not used for calculation.
It is also possible to use a neighboring cell for normalization purposes. This figure shows typical FRA raw data from within R one E ES cells. The graph depicts the unbleached region of the nucleus, which deteriorates on top the bleached region, which shows recovery kinetics at the middle and the background noise level, which is minimal in this case.
On the bottom, results are shown for three proteins, HP one left, H one O middle, and H one E.Right. This figure shows normalized and average data of the FRA curve seen earlier. Note, the slower recovery of H one GFP compared with HP one GFP.
Also, the H one EGFP variant is slower than the H one OGFP variant. Mobile and immobile fractions are indicated for HP one. This figure shows representative raw data of a similar F wrap experiment comparing hetero chromatin to U chromatin of HP one GFP in R one ES cells.
This figure shows normalized and average data of the F wrap curves seen earlier. Note, the slower recovery of hetero chromatin compared with U chromatin mobile and immobile fractions are indicated for U chromatin. This figure shows representative raw data of the flip curve of H one OGFP.
This figure shows normalized and average data of the flip curves seen earlier Once mastered. This technique can be done in less than a minute for each cell if it is performed properly, and therefore an entire experiment can be readily completed within just a few hours. However, experiments should be repeated on different days.
