This protocol makes it possible to isolate high-quality adipose progenitor cells from mouse epididymal adipose tissue for sensitive downstream analysis. This technique yields highly viable adipose progenitor cells and has been validated in a recent single-cell RNA sequencing study. After euthanizing the male mouse, locate the epididymal fat pad and pull on it gently with blunt forceps to deliberate it.
Use scissors to remove the testes and incubate the epididymal fat pad in five milliliters of HBSS supplemented with 3%BSA in a 50 milliliter conical tube for 15 minutes. After the incubation, centrifuge the tube at 150 x g for seven minutes at four degrees Celsius. Remove the floating epididymal fat pad from the conical tube and finely mince it with clean scissors.
Add 200 milliliters of 2%collagenase solution to 3.8 milliliters of HBSS in a 13 milliliter culture tube. Then add the minced tissue and incubate it in a rotating incubator at five RPM for one hour at 37 degrees Celsius. Transfer the entire contents into a 50 milliliter conical tube and add 10 milliliters of neutralization medium.
Mix gently by inverting the tube two to three times. Prepare another 50 milliliter conical tube fitted with a 70 micrometer cell strainer and filtered the digested tissue into the new tube. Transfer the flow through to a 15 milliliter tube and centrifuges at 350 x g per 10 minutes at four degrees Celsius.
Carefully remove the supernatant and resuspend the cell pellet with five milliliters of DPBS. Then centrifuge the tube for another 10 minutes. Carefully remove the supernatant and add 50 microliters of flow cytometry buffer.
Mix the cells by pipetting gently and keep them on ice. The total volume of the cells in the flow cytometry buffer will be approximately 100 microliters. After mixing the cells, transfer 54 microliters of the cell suspension into a 1.7 milliliter microcentrifuge tube.
Add six microliters of FCR blocking reagent and pipette gently to mix. Then incubate the tube at four degrees Celsius for 10 minutes. Keep any leftover cells on ice to use as unstained control.
During the incubation, prepare an antibody cocktail by combining 11 microliters each of anti-Sca1-APC, anti-Ter119-FITC, anti-CD31-FITC, and anti-CD45-FITC antibodies in a microcentrifuge tube. Gently pipette the mixture and keep it on ice protected from light. After the incubation with FCR blocking reagent, add 40 microliters of the antibody cocktail and mix well with gentle pipetting.
Incubate the cells at four degrees Celsius for another 10 minutes. Add 500 microliters of flow cytometry buffer supplemented with one microgram per milliliter DAPI into the stain cells. Mix well and filter the cells using a five milliliter test tube with a cell strainer snap cap.
Then keep the cells on ice. Add 500 microliters of flow cytometry buffer to the unstained cells and mix by pipetting gently. Use a five milliliter test tube with a cell strainer snap cap to filter the cells and keep them on ice.
Transport the stained cells and unstained control to the FACS analysis or sorting instrument. Identify and isolate the APC-positive, FITC-negative, DAPI-negative population using the gating strategies described in the text manuscript. Use the unstained controls to aid in setting gating parameters.
After exclusion of debris and doublets using FSC-SSC plots, viable cells were gated followed by the selection of the APC-positive, FITC-negative population. DAPI, APC and FITC gates were drawn based on the unstained control. The following results show the isolation of adipose progenitor cells from four-month-old male FVB mice.
After one hour of sorting, the quality of isolation was quantitatively evaluated by flow cytometry analysis, which demonstrated that the cells maintained high viability and purity. Sensitive downstream analysis can be performed after this protocol such as single-cell RNA sequencing, quantitative real-time PCR and flow cytometry. This will enable further characterization of the adipose progenitor cells.
Adipose progenitor cells are heterogeneous populations that require further single-cell studies. This method makes it possible to isolate the cells suitable for single-cell studies.
