The overall goal of this procedure is to isolate sub-populations of pericytes from skeletal muscles using FACS. This method can help answer chief questions in the field of pericyte biology, such as what is the biological difference between type I and type II pericytes? The main advantage of this protocol is that it allows simultaneous isolation of type I and type II pericytes without use of NG2 dsRED transgene.

The applications of this protocol are not limited to skeletal muscles. It may also be used to isolate pericytes from other organs, such as the brain. To begin, first obtain the desired tissue from a nestin-GFP transgenic mouse, and then wash the tissue twice in ice-cold PBS containing antibiotics.

Then transfer the samples to a sterile 10-centimeter plate. Next, using sterile scissors and blades, cut the muscles into fine pieces of one to two millimeters cubed. If needed, add a small amount of DMEM to prevent the tissue from drying out.

Pipette the tissue up and down through a 10-millimeter serological pipette to disrupt it mechanically. Then, add fresh digestion solution, DMEM supplemented with 0.2%Type 2 collagenase, to the mixture, and incubate the sample at 37 degrees Celsius for two hours at 35 RPM. Following two hours of incubation, titrate the mixture with an 18-gauge needle and centrifuge the resulting suspension at 500 x g for five minutes.

Then, re-suspend the pellet in 0.25%trypsin-EDTA and incubate the sample at 37 degrees Celsius for 10 minutes. Next, add 10 milliliters of DMEM supplemented with 20%FBS, and centrifuge the sample at 500 x g for five minutes. Once pelleted, re-suspend the cells in 10 milliliters of red blood cell lysis buffer and spin the suspension at 500 x g for five minutes.

Discard the supernatant and re-suspend the pellet in 10 milliliters of sorting buffer. Then, filter the mixture through a 40-micrometer cell strainer, and centrifuge the obtained single-cell suspension at 500 x g for five minutes once more. Re-suspend the pelleted cells in one milliliter of sorting buffer, count them with the hemocytometer, and further dilute to obtain the suspension of five times 10 to the sixth cells per milliliter.

Prior to cell sorting, prepare controls and stain the samples as described in the text protocol. Turn on the sorter and the software, and when asked, scan and insert a 100-micron sorting chip. Then, load automatic setup beads to perform an automatic setup.

Once the automatic setup is completed, open the Experiment tab, and click on Blank Template. Next, in Measurement Settings, enter DAPI for FL1, Nestin-GFP for FL2, and PDGFR-beta for FL3, and uncheck the boxes for the remaining channels. Activate 405, 488, and 561 lasers, and click on Create New Experiment.

Then, click on the Start Compensation Wizard option, and follow the Compensation Wizard software commands. After loading the unstained control, click on Start, and select Detector Threshold Settings to set the sensor gain of the FSC and BSC detectors, and to visualize the population on the scale. Adjust the gain levels of the fluorescence channels to position the negative population on the left side of the histogram, and record data by clicking the Record button.

When asked by the software, load the single-color control, and record data by clicking Start and Record. Then, gate positive population in the histogram and click Next. To perform compensation, open Compensation tab, select Calculate Matrix, then open Calculate Compensation Settings panel and click Calculate.

Once the calculation is completed, click Finish to close the Compensation Wizard. Now that the compensation has been performed, load the FMO control for PDGFR-beta staining. Click Start.

In the All Events plot, double-click on Gate A to generate a daughter population plot. Then, assign y-axis of the daughter plot to DAPI, and draw Gate B comprising only viable cells. After creating another daughter plot, assign its x-axis to FSC-H, and y-axis to FSC-W, and draw Gate C around the population of single cells.

For Gate C, generate a daughter plot of PDGFR-beta versus Nestin-GFP. Then, click the Record button to record data. Finally, load the sample, record the data, and then click on Pause to preserve the sample.

Set the gating boundaries for PDGFR-beta positive and Nestin-GFP positive cells, and create gates for Type I and Type II pericytes populations. Here shows the gating boundaries for Type I and Type II pericytes in PDGFR-beta-PE-FMO Control. In the Sorting Method tab, select Two Way Tubes and assign type I and type II pericytes to the left and right collecting tubes.

Place 15-milliliter collecting tubes pre-filled with five milliliters of sorting buffer on the collection stage, and click the Load Collection button. Click the Resume button to run the sample, and Start Sort to collect the cells. Once sorted, centrifuge the cells at 500 x g for five minutes.

Re-suspend the resulting pellet in one milliliter of pericyte medium, and count the cells in a hemocytometer. Seed pericytes of both types in a 24-well plate containing coverslips pre-coated with poly-D-lysine at a density of approximately one times 10 to the fourth cells per centimeter squared. Culture the cells in 0.5 milliliters of pericyte medium for three days at 37 degrees Celsius and 5%carbon dioxide.

After 3 days of incubation, examine pericyte morphology, and, under a fluorescent microscope, assess their endogenous expression of nestin GFP. Then, replace the pericyte medium with 0.5 milliliters of adipogenic or myogenic medium to initiate cell differentiation. Change the medium every two to three days.

After 14 days of differentiation, stain the pericytes to visualize perilipin and s-myosin and image the cells under the fluorescent microscope. Presented here are microscopic images of type I and type II pericytes that, following the isolation, were grown in pericyte medium for three days. Type I pericytes were characterized by the round cell bodies with short projections and lack of the endogenous GFP expression.

Type II pericytes had small cell bodies with long and thin processes, and had shown endogenous GFP expression. When both cell types were cultured in a dipogenic medium, type I pericytes differentiated into adipocytes, as evidenced by the pronounced expression of an adipocyte marker, perilipin. On the contrary, Type II pericytes did not respond to a dipogenic differentiation.

Finally, pericytes of both types revealed distinct responses to myogenic medium. Type I pericytes did not undergo differentiation into myocytes, while type II pericytes formed myotubes, as evidenced by s-myosin-positive staining, confirming their myogenic activity. While attempting this procedure, it's important to include FMO controls to determine the gating boundaries.

After watching this video, you should have a good understanding of how to isolate type I and type II pericytes from skeletal muscle by fluorescence-activated cell sorting.