This procedure relies on plants expressing GFP in specific cell types, which can then be isolated from the rest of the plant cells using fluorescence activated cell sorting or facts. This example uses two marker lines expressing in specific cell types of the Arabidopsis. Aliana root seedlings are grown in phyto trays or on agar plates.
Their roots are harvested and treated with cell wall digesting enzymes. After one hour, the solution is filtered to remove large debris. The solution is then centrifuged and the supernatant is removed.
GFP positive protoplasts are subsequently separated by fax. The sorted material can be used for cell type specific analysis such as genome wide transcriptional profiles. Hi, I'm Basian Barman from the laboratory of Ken Birnbaum in the Department of Biology at New York University.
Today we will show you a procedure for fluorescence activated cell sorting of plant protoplasts. In this example, we will be sorting two specific cell types in the Arabidopsis route. We use this procedure in our laboratory to study cell type specific transcriptional profiles.
So let's get started. To begin this procedure, grow wild type seedlings as well as seedlings that have cell type specific expression of GFP. The scarecrow promoter driving GFP marks the root endodermis and the quiescent center in one group of seedlings.
And in another group, the root quiescent center is marked by the walks five promoter driving GFP. The seedlings are grown hydroponically and FIA trays on top of a nylon filter, which lets the roots grow through into the growth medium. Alternatively, plants can be grown on top of a nylon mesh and vertically positioned 1%agar plates.
In addition to helping in the harvest of the roots, the filters allow the seedlings to be transferred on mass to new phyto trays or agar plates where they can be supplemented with a catalyst of interest. This may be done to study cell type specific transcriptional responses to nutrients, hormones, or stress treatments. Check under the fluorescence microscope to verify that the fluorescent marker is expressed as expected.
Make sure the expression pattern of the marker is consistent under the applied treatment conditions since it can change as a result of treatment. Proceed to harvest and prepare protoplasts. Start by preparing the protoplasts solution.
Combine 1.25%weight per volume cellulase 0.3%weight per volume, maser zyme 0.4 molar D mannitol 20 millimolar, MES, and 20 millimolar KCL in demineralized water and adjust the pH to 5.7 with one molar tris HCL at pH 7.5. This solution will be slightly turbid. Then heat the solution to 55 degrees Celsius for 10 minutes.
The solution will turn clear, let it cool down to room temperature and then add 0.1%weight per volume BSA 10 millimolar calcium chloride, and five millimolar beta me capto ethanol harvest one week old seedlings from one phyto tray. In the case of the scarecrow GFP line or eight plates of four day old walks 5G FP seedlings. Scrape the roots off the nylon mesh with a scalpel, then deposit them into a container with a protoplasts solution.
Use approximately 10 milliliters of protoplasts solution per 1500 seedlings. Shake the flasks gently at 75 RPM at room temperature for one hour. A longer incubation time may increase the protoplasts yield, but will also add to the effect of protoplasts itself on gene expression.
Now that the protoplasts are released, use a 40 micrometer cell strainer to filter the solution and divide the protoplasts suspension between conical 15 mil tubes. It is important to generate clean protoplasts suspension without too much debris to prevent clogging of the facts and to minimize the time needed to sort the cells. Centrifuge the tubes in a swing bucket centrifuge at 500 G for 10 minutes at room temperature.
Note that the centrifugation speed depends on the constitution of the plant section yielding the protoplasts and the amount of cell debris produced during enzymatic treatment. After centrifugation, remove most of the supernatant and gently resuspend the pellet containing the protoplasts in the small volume of remaining protoplasts solution. Finally, count the protoplasts using a hemo cytometer and determine their density to calculate the yield and decide on the facts run parameters.
After counting, inspect the protoplasts. Check their integrity, the level of GFP expression and the content of contaminating cell debris. Next, proceed to fax.
If not proceeding directly to fax. Protoplasts can be washed, resuspended and stored in an incubation solution such as proto plating solution without the added enzymes or W five solution. Turn on the cell sorter and the workstation computer.
Here we use a fria manufactured by Becton Dixon and Company. Use one XPBS as a sheath fluid and run the fluidic startup procedure. Install a 100 micrometer nozzle and set a 20 PSI sheath pressure.
Set up a stable flow stream and calibrate the drop delay. Note that calibration is not shown in this procedure. Protoplasts suspensions with a density as high as 10 million cells per mil can be successfully sorted.
In order to prevent sedimentation of the protoplasts, use the sample agitation option on the facts. If clogging of the facts is an issue, there are three possible shooting steps. First, perform a sample line back flush.
Second, dilute your protoplasts suspension to reduce the density. Third, clean up the protoplasts solution by repeating the filtration step after centrifugation and resus suspension. Just four parameters are used to distinguish GFP positive protoplasts from GFP negative protoplasts and cell debris after excitation by a 488 nanometer laser forward scatter is an indicator of particle size, side scatter as an indicator of particle granularity.
Emission at 530 nanometers as a measure of green fluorescence and emission at 610 nanometers as a measure of red spectrum autofluorescence. Start by setting up a dot plot for forward scatter versus side scatter. Apply the voltage setting so that the measured events are centered in the plot.
GFP positive PROTOPLASTS can be distinguished by their increased ratio of green to red emission compared to events with autofluorescence. Only set up a dot plot with green fluorescence versus red spectrum autofluorescence. Apply voltage settings in such a way that the measured events give rise to a single diagonal population in the plot.
When looking at a wild type protoplasts suspension, GFP positive PROTOPLASTS should produce a clear population of green fluorescent events never seen in wild type samples. Set compensation constraints to adjust for spectral overlap between green fluorescence and red spectrum autofluorescence. Using A GFP positive PROTOPLASTS suspension, proper compensation constraints settings will allow for better separation of the GFP positive protoplasts from the non GFP protoplasts and debris set up a gate to identify GFP positive events.
It is advisable to take along non GFP PROTOPLASTS each time you prepare a sorting experiment to help define the gate boundaries. Finally, set a forward scatter threshold cutoff in order to leave small debris out of the analysis. The visualization of GFP positive events in the forward scatter versus side scatter plot will help in determining where the threshold should be set.
The parameters set up in this initial facts run can be reused for future sorts. Slight adjustments will be required for day-to-day use of the facts. For RNA extraction prepare collection tubes containing RNA extraction buffer at most use 100 microliter sort volume to 350 microliter extraction buffer as a guide.
20, 000 sorting events yield a total sort volume of approximately 100 microliters. Now that the fax parameters and operating modes are set and the collection tubes are ready, start sorting the protoplasts when the sort is complete. Mix the sample collection tubes as cell suspension can pool at the top as few as 500 sorted events can be used for microarray analysis following RNA extraction and CD NA amplification.
Here we use the RNEZ micro extraction kit, the WT ovation PICO RNA amplification system and the fl ovation CD NA biotin module V two. Here are typical fax results for protoplasts derived from non GFP Scarecrow, GFP and W 5G FP seedlings 100, 000 events are presented in each dot plot of green fluorescence on the x axis versus red fluorescence. On the Y axis, the events falling within the GFP sorting gate are highlighted green.
This is a summary of the typical protoplasts yield from the seedlings expressing either scarecrow GFP marking the root endodermis and quiescent center or walks 5G FP marking the root quiescent center. There are fewer cells in the root quiescent center compared with the root endodermis. Therefore, despite starting out with more protoplasts, the yield of GFP expressing cells is smaller in the walks 5G FP sort compared with the scarecrow GFP sort shown here are typical RNA extraction results of triplicate samples.
Each sample corresponds to the RNA extracted from 10, 000 events. The extracted RNA is analyzed on a bioanalyzer showing the ribosomal peaks for the scarecrow GFP samples on top and the walks 5G FP samples below the extracted RNA can be further used for microarray analysis. This scatter plot of log two expression levels demonstrates the similarity between two replicates.
We've just shown you how to perform fluorescence activated cell sorting of plant protoplasts for cell type specific analysis. This technique is applicable to many different plant tissues and species. Good yields with low debris content and healthy protoplasts will give better downstream results in transcriptional as well as other analyses.
Also remember that it's important to keep growth conditions of the plant material identical for comparison between sorted samples. So that's it. Thanks for watching and good luck with your experiments.
