Flow cytometry analyzer

We collected purified peripheral blood mononuclear cells (PBMCs) from the peripheral blood samples of healthy donors and colon cancer patients by using a Ficoll-Paque (Haoyang Biotec, #LTS1077) differential density gradient centrifugation process according to the manufacturer’s instructions. The proportions of γδT cells in PBMCs were determined by a flow cytometry analyzer (Beckman Coulter, Inc.).
Primary γδT cells were isolated from colon cancer tissue samples and neighboring noncancerous tissue samples by enzymatic digestion.^28,29 Briefly, the tissue samples were stored in a 0.9% aqueous sodium chloride solution (Baxter Healthcare (Shanghai) Co., Ltd., #A6E1307) at 4°C, washed with PBS (HyClone, #SH30256.01B), cut into small fragments, and incubated for 2.5 h at 37°C in a 2 mg/mL type IV collagenase (Sigma-Aldrich, #V900893) solution. After digestion, disaggregated cells were filtered through a 40 μm strainer (SPL Life Sciences Co., Ltd., #93040) and harvested at 400 g for 5 min. The proportions of γδT cells among the disaggregated cells were determined by a flow cytometry analyzer (Beckman Coulter, Inc.).

After the isolation of USCs, we used the Alizarin Red Staining, Oil Red O staining, and Alcian blue staining to verify their multilineage differentiation potential. For osteogenic differentiation, USCs were incubated with an osteogenic induction medium (Gibco). On day 21, after the induction, the cells were stained with Alizarin Red S (Sigma, USA) for observation. For adipogenic differentiation, USCs were incubated with an adipogenic medium (Gibco). On day 21, after the induction, USCs were stained with Oil Red O (Sigma) for observation. For the chondrogenic differentiation, USCs were incubated with a chondrogenic medium (Gibco). After 21 days of induction, allicin blue staining (Sigma) was performed for observation. We also performed the flow cytometry analysis to detect the stem-cell surface markers (CD44, CD73, CD90, CD105, CD31, CD34, and CD45). The USCs were incubated with these antibodies (BD, USA), washed with PBS, and analyzed with a flow cytometry analyzer (Beckman, USA).