T75 culture flasks

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

The PDLSCs were seeded in T75 culture flasks (BD Pharmingen) and maintained in 5% CO₂ incubator at 37 °C for 24 h until they reached 70–80% confluency. Then, the PDLSCs (n = 4) were exposed to ASA [0 (control), 200, 500, 1000 μM] in osteogenic media containing 10% FBS, 1% l-alanyl-l-glutamine, 100 nM dexamethasone, 10 mmol/L β-glycerol phosphate and 0.2 mM of ascorbic acid for 21 days. The media was changed every 3 days. Total RNA was extracted at day 21, using the RNAeasy Mini kit (Qiagen).

All experiments involving human material were performed under ethical approval from the Swedish Ethical Review Authority (register number 2018/97‐31) and in accordance with ethical standards at the Linköping University and Swedish and European regulations. Skin was obtained from healthy female patients undergoing routine breast reduction surgery, and all material was de‐identified.
Briefly, human keratinocytes were isolated through mechanical dissection and incubated in 15 ml Dispase (Gibco, Life Technologies; 16.7 mg/ml, 1.04 U/ml) for 18 h at 8°C. The epidermis was transferred to 2 ml ethylenediaminetetraacetic acid (EDTA; 0.02%) and 2 ml trypsin (Invitrogen, Life Technologies) to dissociate cells. Cells were washed in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS) and centrifuged at 400× g for 5 min. The cells were seeded in T‐75 culture flasks (Falcon) in complete keratinocyte serum‐free medium (KSFM; Invitrogen, Life Technologies) supplemented with 25 µg/ml bovine pituitary extract (BPE) and 0.2 ng/ml EGF as provided by the manufacturer, and 50 U/ml penicillin and 50 mg/ml streptomycin (Gibco BRL, Life Technologies).