Large-size fat particle sorting

Large-size fat particle sorting is widely used to isolate cells up to 200 μm in diameter. The use of a dedicated flow cytometer equipped with a large nozzle is required, and the ability to use a low-pressure during cell sorting to reduce shear stress is also required. Conventional flow sorters are not designed for adipocyte sorting due to their small nozzle size and high shear force, which may cause lipolysis during cell sorting. Single-cell flow sorting will allow greater insight into adipocyte heterogeneity by identifying gene expression, protein composition, and metabolic signatures at the single-cell level.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Isolation of fat cells

1.1 For each sample, at room temperature, collect 1 g of white adipose tissue (WAT) in 50 mL conical tubes, containing 25 mL of adipocyte culture medium.

1.2 Place wet water into a 6-well plate at room temperature and gently cut into 2 to 5 mm pieces with a surgical scalpel.

1.3 Transfer all fragments to a 50 mL conical test tube containing 5 mL of 3 mg/mL collagenase solution, preheated to 37°C.

1.4 Incubate at 37 °C for 30-40 minutes, rotating the tube manually every 5 minutes.

1.5 Pass the partially digested water suspension through a 100 μm cell strainer into a 50 mL conical tube. Partially digested water often contains a mixture of small tissue fragments and an opaque cell suspension. The appearance of clear oil is usually a sign of overdigestion and should be avoided. Using a 100 µm cell strainer may result in loss of large adipocytes.

1.6 Wash the filter with 10 mL adipocyte culture medium at room temperature.

1.7 Gently mix the 50 mL tube containing the digested tissue and incubate at room temperature for 10 minutes. During this gravity wash, fat-rich fat cells will float to the top of the tube.

1.8 Collect 1-2 mL of the top layer with a 1 mL pipette and transfer to a 15 mL conical tube containing 10 mL of adipocyte culture medium.

1.9 Mix and incubate the 15 mL test tube according to step 7, then collect 1 mL of washed adipocytes, and then transfer to a fresh 15 mL test tube containing 10 mL of adipocyte culture medium.

1.10 Repeat the wash once and transfer the last 1 mL of the suspension to an empty tube.

2. Label and fix adipocytes

2.1 Add the following to the washed adipocytes and mix gently: 250 μL10 μM BODIPY-C12; 10 μL 100× calcein; 2 μL 10 mg/mL Hoechst 33342.

2.2 Incubate 15-30 min at 37 °C with gentle agitation and protection from light.

2.3 Place the tube on the bench for 5 min to allow adipocytes to float. Remove most of the medium beneath the adipocytes using a 200 μL pipet tip or a needle.

2.4 Add 10 mL adipocyte medium to the adipocytes and incubate at room temperature for 10 min.

2.5 This will dilute the labeling mixture and stop the reaction. This washing step can be repeated once or twice to ensure that fluorescent tracers are completely removed from the medium.

2.6 Collect adipocytes (0.5-1 ml) from the top fraction and transfer to a 15 mL conical tube containing 9 mL PFA.

2.7 Incubate 15-20 min at room temperature, protected from light.

2.8 Wash three times with PBS using the gravity method.

3. Analyze and sort adipocytes

3.1 Open the sample valve and sheath valve to prime the tubing connecting the Luer-lock syringe to the flow cell until the tubing fills with sheath fluid (PBS).

3.2 Adjust the rotation of the Luer-lock syringe such that it rotates the syringe 180° once every 2-3 s.

3.3 Load adipocytes into a Luer-lock syringe at a concentration of 1000-2000 cells/mL.

3.4 Select “refill syringe” and follow the instructions.

3.5 Analyze a total of 5,000-10,000 objects and then press Abort.

3.6 Select which region from the Histogram to sort from (found under the Sorting Gate section in the Acquire/Dispense window).

3.7 Choose a number of adipocytes from the selected region to sort out into a receptacle (e.g., a 1.5-ml microcentrifuge tube).

3.8 Fill the wells of a 96-well plate with a small amount of PBS (30-50 μL) and place the plate on the plate holder.

3.9 In the FlowPilot main menu, under the Setup tab, go to Plates and then Select Calibrated Plate.

3.10 Set the coincidence mode to Pure, no doubles.

3.11 Press Fill Plate.

4. Analyze adipocytes by microscopy

4.1 Transfer each sorted adipocyte in 50-100 μL medium onto a microscope slide for imaging. Image adipocytes using one of these two alternative methods:

a. Invert the slide, keeping the medium droplet attached to the glass surface by surface tension. Adipocytes will float to the top and can be imaged using an inverted or an upright microscope.

b. Transfer the drop (in a final volume of <100 μL) to the slide and gently cover with a circular coverslip. To avoid cell lysis due to the force of suction, place a thin spacer between the slide and coverslip.