Singlequots supplements and growth factors

Human embryo fibroblasts (HFs) (OUMS-36T-1) were purchased from JCRB Cell Bank (Osaka, Japan) and cultured in a 37 °C and 5% CO₂ incubator with DMEM (Nacalai Tesque) supplemented with 10% FBS (Gibco) and 5% antibiotic-antimycotic solution 100× (Gibco). The medium was changed every two days. HFs were passaged when they became sub-confluent (approximately 70% confluent).
Normal bone marrow-derived human mesenchymal stem cells (hMSCs) were purchased from Lonza and cultured in a 37 °C and 5% CO₂ incubator with hMSC basal medium containing SingleQuots™ Supplements and Growth Factors (PT-3001, Lonza). The medium was changed every two days. HFs were passaged when were sub-confluent (approximately 80% confluent). hMSCs (passage 3–5) were used in differentiation experiments.

Our in-vitro culture model of magnum explant has been adapted from Kasperczyk et al.^{25 (link)}. Briefly, magnums from eight laying hens were collected by dissection after slaughtering and kept in saline solution for about 30 min at room temperature before use. For each culture, one piece of magnum tissue was washed twice in Phosphate Buffered Saline (PBS) plus penicillin (100 U/ml)/streptomycin (100 μg/ml) and subsequently finely cut in small pieces of 1 mm of diameter. Between two and three explants were cultured in each well of a 24-well culture plate and incubated with E2 and/or P4 at 37 °C in Gold Keratinocyte growth Basal Medium (KBMTM, Lonza, ref 00192151), supplemented with SingleQuotsTM Supplements and growth factors (Lonza, ref 00192152) without serum. To determine the amount of secreted chemerin within the culture medium, explants were subjected to 48 h of stimulation. At the end of the experiment, the conditioned culture medium was kept at – 20 °C before use for chemerin assays. Eight independent cultures were performed.

The BCi-NS1.1 cell line was derived from human airway basal cells via expression of a retrovirus expressing human telomerase and was a kind gift of Dr. Ronald Crystal, Cornell Medical Center [19 (link)]. BCi-NS1.1 cells were cultured in BEBM^TM Bronchial Epithelial Cell Growth Basal Medium (Lonza, cat#CC-3171) with SingleQuots^TM Supplements and Growth Factors (Lonza, cat#CC-4175). The human pulmonary epithelial cell line, A549, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, CCL-185). A549 cells were propagated in Dulbecco’s modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 80 µg of gentamicin/mL.

Human umbilical vein endothelial cells (HUVECs) were maintained in endothelial cell growth basal medium-2 (EBM-2; Lonza, Walkersville, MD) supplemented with SingleQuots^TM Supplements and Growth Factors (Lonza; USA, MD). Matrigel (Corning, Flintshire, UK) was diluted with serum-free EBM-2 medium and used to coat in 96-well plates at 37 °C for 1 h. HUVECs were seeded at a density of 1 × 10⁴ cells per well of a 96-well plate in EBM-2 or conditional medium. The number of tube formations was imaged and analyzed using phase contrast microscopy and ImageJ 1.53e software (National Institutes of Health).