Singlequots kit

NHEKs were purchased from Lonza (Clonetics Lonza, Walkersville, MD, USA) and maintained in keratinocyte growth medium (Keratinocyte Basal Medium (KBM) supplemented SingleQuots^TM Kit; Lonza, Basel, Switzerland) at 37 °C in a humidified atmosphere containing 5% CO₂. NHEKs were synchronized as follows: NHEKs were grown in growth medium until the cells reached 70–80% confluency. The medium was then replaced with a serum-rich medium (KBM supplemented with 50% horse serum (Gibco)) and incubated for 2 h. The cells were collected for analysis of the mRNA and protein expressions at the indicated times. For UV irradiation, the cells synchronized in a serum-rich medium for 2 h were treated with 20 mJ/cm² UVB, using BIO-SUN UV-H (VILBER LOURMAT, Collégien, France) in PBS solution, and the cells were then cultured back in fresh KBM. Immediately after the UVB irradiation, 10 ng/mL of human synthetic TIMP3-peptide (Abcam, Cambridge, UK) or Camellia sinensis leaf (CSL) extracts at different concentrations in KBM were used for the treatment. The CSL extracts used in this study were obtained from a new green tea tree species named Jangwon No.3 [19 (link)] that was cultivated by AmorePacific Corporation. The cells were harvested at the indicated times for analysis of the mRNA and protein expressions of each gene.

Human retinal microvascular endothelial cells (HRMEC), donated by Dr. Jian-Xing Ma from the University of Oklahoma, were cultivated in an EBM endothelial cell growth medium (Lonza Bioscience, Basel, Switzerland) supplemented with an EGM endothelial cell growth medium SingleQuots kit, including hEGF, hydrocortisone, GA-1000, BBE, ascorbic acid, and FBS (Lonza Bioscience) in a humidified incubator at 37 °C and 5% CO₂. The cells were treated with either CoCl₂ (200 $\mathsf{\mu }$ M or 300 $\mathsf{\mu }$ M) or the complete routine medium once they reached confluency of 70–80%. Forty-eight hours later the cells were stained with anti-α_v for further assessment. Briefly, the cells were fixed in 4% PFA and blocked in 10% horse serum. Then, they were stained with anti-α_v integrin (SC9969, Santa Cruz, CA, USA) primary antibodies. The secondary antibody used was anti-mouse Alexa Fluor 594 (A11005, Invitrogen, Waltham, MA, USA) and 4′6′-diamino-2-phenylindole (DAPI, H-1500, Vector Laboratories, Newark, CA, USA) was used for nuclei visualization. Using HRMECs cultured as described above, ASL exosome cellular uptake was also evaluated. After confluence reached 70–80%, either CoCl₂ or the regular routine medium was applied for 24 h. ASL exosomes were then added, and cells were incubated for a further 24 h. After a total 48-h incubation they were fixed and stained with anti-α_v integrin antibodies.

Human hepatoma cell line HuH-7, Baby hamster kidney cell line BHK-21 and Aedes albopictus cell line C6/36, purchased from the Japanese Collection of Research Bioresources (Japan) and the American Type Culture Collection (ATCC, Manassas, Virginia), were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone, Logan, UT). Human umbilical vein endothelial cells (HUVECs) were purchased from the Bioresource Collection and Research Center (BCRC) of Taiwan and maintained in endothelial Basal Medium-2 (EMB-2, Lonza, Walkersville, MD) supplemented with 10% FBS, and SingleQuots™ Kit (Lonza, Walkersville, MD). All cells were cultured at 37 °C in a 5% CO2 atmosphere, except for C6/36 which were cultured at 28 °C. Four serotypes of dengue viruses, DENV 1 (local Taiwan strain 8700828), DENV 2 (16681 and local Taiwan strain 454009 A), DENV 3 (local Taiwan strain 8700829), and DENV 4 (local Taiwan strain 59201818) were propagated in C6/36 cells as previously described^{52 (link)}. To prepare high titers of DENV, cell-free supernatants were concentrated by Macrosep® Advance Centrifugal Devices (molecular weight cutoff of 30 kDa; Pall Corp., Port Washington, NY) at 6000 × g at 4 °C and stored below −70 °C until use.

Normal human bronchial epithelial cells (HBECs; CC-2540; Lonza) were expanded twice with growth medium (500 ml BEGM medium [CC-3171; Lonza] and one SingleQuots kit [CC-4175; Lonza]) in T75 flasks to obtain passage 2 (P2) cells, which are >99% p63⁺ basal cells. ALI cultures using P2 cells were performed as previously described (Danahay et al., 2002 (link)). The packaged virus for the pooled shRNA library was added within 1 h after cell seeding at an MOI of 1. The cells were cultured in differentiation medium (250 ml BEGM medium, 250 ml DMEM [11965092; Thermo Fisher Scientific], and one SingleQuots kit lacking triiodothyronine, with a final concentration of 50 nM all-trans retinoic acid) on both apical and basal sides of Transwells for the first 7 d. Medium was removed from apical side, and cells were cultured for another 2 wk under ALI conditions. At day 21, cells were harvested with 0.05% Trypsin-EDTA (25300054; Thermo Fisher Scientific), fixed, and sorted based on FOXJ1 and ITGA6 signals following the modified MARIS protocol. The gDNA from sorted cells was extracted with RecoverALL Total Nuclei Acid Isolation kit (AM1975; Ambion) for NGS analysis.

HRMECs were subscribed from Cell Systems Corporation (USA) and grown in Endothelial Basal Medium-2 (Lonza, Switzerland) supplemented with SingleQuots kit (Lonza). HRMECs up to passage 8 were grown at about 80% confluency. After transfection, the cells were starved in a minimal medium overnight to eliminate the effects of growth factors. Then cells were stimulated by 10 ng/mL Recombinant Human VEGF₁₆₅(R&D Systems, USA).