Round bottom tube

Frozen PBMC vials were removed from liquid nitrogen storage tanks and placed into a 37 °C water bath. Once samples were thawed and removed from the water bath, the vials were wiped down with 70% isopropyl alcohol and the cell suspensions transferred using plastic transfer pipettes (Fisher Brand) to a 15 mL round bottom tube (Corning). Roswell Park Memorial Institute Medium (RPMI [Gibco]) with 10% Fetal Calf Serum (FCS [Omega]), 1% L-glutamine (Gibco), 100 U/mL Penicillin-Streptomycin (Gibco), 12.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES [Gibco]) buffer was added dropwise to each round bottom tube to gradually dilute the cell suspension. Between dropwise additions of culture medium, the cell suspensions were agitated to ensure homogeneity. Cells were centrifuged at 300g for 10 min at room temperature, then resuspended in 1 mL of 10% FCS-RPMI. Cell suspensions were counted using a Z2 Particle Counter (Beckman Coulter) and viabilities were assessed using 1:4 dilution of cell suspension to 0.2% Trypan Blue under a hemocytometer chamber; mean viability of PBMC was 89.6%. Thawed PBMC were divided for DNA extraction and flow cytometry.

The cells after 12–13 weeks of differentiation by the stepwise protocol were washed with PBS and treated with Collagenase mix solution at 37℃ for 7 min. Collagenase mix solution is composed of DMEM supplemented with 0.05% Collagenase H (Meiji Seika Pharma, Tokyo, Japan) and 0.5% Collagenase G (Meiji Seika Pharma). Accutase was added to the solution and incubated at 37 °C for 10 min. The solution was neutralized with DMEM + 10% HS and centrifuged at 380 × g, 4 °C for 7 min. After removal of the supernatant, the cells were resuspended in DMEM + 10% HS and filtered through a 40-µm Cell Strainer (Corning). To remove debris and dead cells, density gradient centrifugation was performed using Optiprep (Serumwerk Bernburg, Bernburg, Germany) in DMEM + 10% HS (ρ = 1.11 g/mL), in accordance with the manufacturer’s protocol. The fraction of living cells was resuspended in HBSS + 1% BSA and cytometry was performed. The cells were incubated with an APC-conjugated anti-CDH13 antibody (5 × 10⁶ cells/mL) on ice for 15 min. Thereafter, they were diluted in HBSS + 1% BSA and centrifuged at 380 × g, 4 °C for 5 min. After removal of the supernatant, the cells were resuspended in HBSS + 1% BSA containing Hoechst 33342 (1:2000, 5 × 10⁶ cells/mL) and filtered with a Round-Bottom Tube with Cell Strainer (Corning). The prepared cells were stocked on ice before the flow-cytometric analysis.