Type 1

This study was approved by the Medical Ethics Committee, Faculty of Dentistry, University of Malaya [Medical Ethics Clearance Number: DF CO1107/0066 (L)] and with the informed consent of the patients. The PDLSCs were isolated from normal and vital tooth. The donors were aged between 18 and 35 years old (n = 3) and the teeth were indicated for extraction for orthodontics treatment purposes. The PDLSCs were isolated by using standard protocols with some modifications [4 (link)]. The PDL tissues were scraped off the root surface using a sterilized scalpel and minced into small fragments prior to digestion in a solution of 1 mg/mL collagenase type I (Gibco, Grand Island, NY) for 30 min at 37 °C.
After neutralization with a 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY), the cells were centrifuged and seeded in T75 culture flasks (BD Pharmingen, San Diego CA, USA) using a culture medium containing KO-DMEM, 10% FBS, 0.5% and 10,000 µg/mL of penicillin/streptomycin (Invitrogen), 1% 1× Glutamax (Invitrogen), and incubated at 37 °C in the presence of 5% CO₂. The medium was replaced every 3 days until the cells reach 70–80% confluency

Tumor samples were dissected into small pieces using scissors. To digest the tissue fragments into single-cell suspension, DMEM containing type I (1 mg/mL, Gibco) and type IV collagenase (1 mg/mL, Gibco) was employed and cultivated at 37°C for 30 minutes. Then, the single-cell suspension was obtained by filtration with a 70 μm cell filter. After being washed twice with D′-HANKS buffer, the cells were cultivated with Fc-Block to avoid nonspecific binding. The following fluorophore-conjugated antibodies (Biolegend) were used for staining analysis: CD3-Percp-cy5.5 (#100218), CD45-APC-Cy7 (#103154), CD8-PE (#100707), CD44-PE-Cy7 (#103030), CD4-FITC (#100510), CD69-BV710 (#104537), and CD62L-BV605 (#104437). Cells were defined by cell surface molecules as follows (1): CD45⁺CD3⁺presents T cells (2); CD45⁺CD3⁺CD8⁺ indicates CD8⁺ T cells (3); CD45⁺CD3⁺CD4⁺ presents CD4⁺ T cells (4); CD8⁺CD69⁺ indicates activated CD8⁺ cells (5); CD8⁺CD44⁺CD62L⁺ presents memory T cells. After being incubated on ice for 30 minutes and washed, the cells were collected for flow cytometry analysis. The final results were expressed as the proportion of various cell subtypes in CD45⁺ cells.