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The overall goal of this procedure is to isolate a population of cell type specific protoplasts from a leaf. This is accomplished by first growing plants that express GFP in specific cell types in the leaf. The second step is to harvest leaves and generate protoplasts using an enzymatic digestion.

Next, the protoplasts are filtered and sorted using fluorescence activated cell sorting or facts. The final step is to confirm the accuracy of the sorting using fluorescence microscopy. Ultimately, the sorted protoplasts can be used for analysis of cell type specific gene expression, proteomic analysis, or metabolic profiling.

Fluorescent activated cell sorting has previously been used successfully for roots, but it's used for isolating leaf cells. Labeled with GFP prior to this method has been limited due to the interference from chlorophyll fluorescence. Visual demonstration of this method is critical as rapid, but careful sample preparation is crucial for minimizing the extensive physiological changes that can occur during cell sorting.

Generally, individuals new to this method will struggle because they will not be aware of how to set up the cell sorter or how to confirm accurate sorting. This method can help answer key questions in the plant systems biology field. The problem with identifying gene regulation networks in a complex multicellular organs such as a leaf, is that different cell types express different groups of genes.

This genetic information is lost in the harvesting analysis of a whole leaf Demonstrating the procedure. Alongside me will be Ellison As and Sanjeev Kuma, who provide technical support for this work in our laboratory. First, fill a nine centimeter round Petri dish with 20 milliliters of solution A containing proto plating enzymes and transcriptional and RNAs inhibitors.

Then harvest the desired leaf material for samples containing more than a single leaf like the sample shown here. Arrange the leaves in up to a 15 leaf stack. Then use a razor blade to cut the leaves into one millimeter wide strips.

Immediately transfer the leaf strips into the Petri dish using a pair of flat tweezers, gently separate the strips and then vacuum infiltrate the samples two times for five minutes each time. Next, place the Petri dish on an orbital shaker set to 90 RPM at room temperature and proto plaster samples for three to four hours. During the incubation, use the tweezers to separate the leaf strips from each other at half hour intervals.

After the digestion, gently filter the protoplasts solution through a 70 micron cell strainer into a 50 milliliter tube. To remove the leaf strips, the strips will be retained. The protoplasts will filter through.

Now use four milliliters of solution A to gently rinse the leaf strips collecting the wash in the 50 milliliter tube. Then transfer the protoplasts suspension into round bottom tubes and centrifuge the protoplasts for five minutes at 300 times G and room temperature. Being careful not to disturb the pellet, gently aspirate off as much of the supine natant as possible, and then wash the protoplasts with five milliliters of solution A under the same centrifuge conditions.

Finally, after removing the supine natant, use a cutoff P 1000 tip to resuspend the PROTOPLASTS in an appropriate volume of solution A for sorting immediately before sorting. Use an uncut P 1000 tip to disaggregate any clumps in the Protoplasts solution. Further dissociate the cells by filtering the protoplasts into a sourcing tube through a 50 micron fillon filter.

Then dilute the cell solution as appropriate to avoid clogging the cell sourcing nozzle. After determining the optimal cell sourcing conditions, identify the GFP positive Proto plus with a 488 nanometer Argonne laser plussing the output from the 580 30 Band pass filter versus the 530 40 band pass filter. Note that the GFP positive protoplasts will appear in the high 530, low 580 population.

With the non GFP Protoplasts in the low 530, low 580 population, the dead dying protoplasts and debris appear in the high 580 population for visualization of the sorted protoplasts by fluorescence microscopy, sort the protoplasts directly into at least eight volumes of solution A after sorting concentrate, the protoplasts by centrifugation protoplasts can be sorted into eight volumes of solution A and collected for visualization on a fluorescence microscope to verify the enrichment. This first image shows an example of an enrichment of the TP 382 cell line, which expresses GFP in the guard cells. Examples of yields obtained using this method both before and after amplification are summarized in this table.

The amounts obtained between 500 picograms to 50 nanograms are sufficient for amplification by the ovation PICO WTA system and subsequent analysis by quantitative PCR next generation sequencing or microarray. This next series of images show various protoplasts cell lines isolated and sorted by the method just described. These first protoplasts were derived from KC 2 74, leaves a transverse section of a KC 2 74.

Leaf shows that the GFP is expressed in the vasculature. These protoplasts were isolated from KC 4 64 leaves and a transverse section of a KC 4 64 leaf shows that GFP expression is found in the A dal epidermis. Finally, these protoplasts were derived from TP 3 82 leaves in TP 3 82 leaves.

The GFP is expressed in the guard cells here. Typical fax acquisition dot plots illustrating the output from the 5 80 30 nanometers versus the 5 30 40 nanometer band pass filters are shown. The sorting gate shown are those typically used during sorting.

This first stop plot of coal four leaf derived protoplasts proto plastered in the absence of inhibitors shows a single low 530, low 580 population. Whereas this dot plot from wild type coal four leaf derived protoplasts proto plastered in the presence of inhibitors demonstrates the shift in the 580 fluorescence that occurs due to the stress and coloring caused by the inhibitors. These dot plots are of leaf derived protoplasts from the KC seven four, KC 4 64, and TP 3 82 lines respectively.

These samples also were pro plastered in the presence of inhibitors showing the GFP to be contained in the high 530 low 580 populations. For this representative, quantitative PCR experiment samples were first obtained for each fraction type. The samples were then amplified using the ovation PICO WTA system and quantitative PCR.

Using GFP specific primers was performed. The values shown are the highest in red and the lowest in green average or single values using actin two as a reference gene are indicated by the horizontal yellow bars. The results a reverification of the optical assessments of the GFP cell content in the sorted fractions.

The high 530 low 580 population exhibited the highest measurable levels of GFP expression. While the high 530 high 580 population exhibited about 1000 fold lower GFP expression, the low 530, low 580 and low 530 high 580 populations exhibited low levels of GFP expression that were comparable to those of the control whole leaf and unsorted protoplasts populations Once mastered, this technique can be done in four to five hours from plant to sorted cell population if it is performed properly. Whilst carrying out this procedure is important to remember to constantly monitor the cell sorter and to recalibrate it if necessary in order to maintain sorting accuracy.

Found this procedure. Other methods such as microray analysis can be performed in order to compare and contrast gene expression levels in different cell types. Now that this technique is developed, we are analyzing gene expression in specific leaf cell types at different developmental stages, and we'll compare this to data from the whole leaf that has been generated in the agronomics project.

After watching this video, you should have a good understanding of how to isolate single cell types of leaves using fluorescence activated cell sorting.