CAY10683 br Reverse transcription polymerase chain reaction
2.2. Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from 1 × 105 cells using TRIzol reagent according to the manufacturer's instructions (Gibco). Total RNA (2 μg) was reverse-transcribed with MMLV reverse transcriptase (Bioneer, Daejeon, Korea) for 1 h at 42 °C with oligo-dT primers. PCR was Cancer Letters 440–441 (2019) 202–210
performed using Taq DNA polymerase (Bioneer). The specific primers used for the RT-PCR assays were 5′-TTCGAAGCCTTTGCTCTGGCAC-3′ (sense), 5′-AGATGGTCAATGCGGCGTCC-3′ (antisense) for IFN-β; 5′-AAGGGCTTCAGTGACCGGCT-3′ (sense), 5′-GGAGTCTTTCTAACGA GCTGACGG-3′ (antisense) for TRAIL; and 5′-CAAGGCTGAGAACGGGA AGC-3′ (sense), 5′-AGGGGGCAGAGATGATGACC-3′ (antisense) for GAPDH. Amplified products were subjected to 2% agarose gel electro-phoresis and photographed using the FluorChem FC2 system (Alpha Innotech, Santa Clara, CA, USA). All results were normalized against
To detect TRAIL protein expression, cells were lysed in sample buﬀer (62.5 mM Tris-HCl [pH 6.8], 1% sodium dodecyl sulfate, 10% glycerol, and 5% β-mercaptoethanol), boiled for 5 min, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and trans-ferred to an Immobilon membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk in Tris-HCl-buﬀered saline containing 0.05% Tween 20 and then incubated with primary anti-bodies against TRAIL (1:1000; R&D Systems, Minneapolis, MN, USA); caspase (Cas)-3, cleaved Cas-3, and Cas-8 (1:1000; Cell Signaling Technology, Danvers, MA, USA); and GAPDH (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary CAY10683 (1:2,000, Santa Cruz Biotechnology), treated with EZ-Western Lumi Pico (Dogen, Seoul, Korea), and visualized using the FluorChem FC2 system (Alpha Innotech).
2.4. Membrane-bound antigen analysis
To confirm whether ASCs express membrane-bound TRAIL (mbTRAIL) or DR5, a TRAIL receptor, ASCs were stained with anti-bodies conjugated with Fluor 488 or allophycocyanin (APC) against mbTRAIL (i.e., mbTRAIL-Fluor 488 antibody) or DR5 (i.e., DR5-APC antibody), respectively (BD Biosciences, San Jose, CA, USA). A total of 5 × 105 cells were resuspended in 0.2 ml PBS and incubated with mbTRAIL-Fluor 488 or DR5-APC antibodies for 20 min at room tem-perature. Fluor 488- or APC-conjugated mouse IgGs were used as the control isotype at the same concentration as the specific primary anti-bodies. The fluorescence intensity of the cells was evaluated by flow cytometry (BD FACSAria III; BD Biosciences), and the data were ana-lyzed using BD FACSDiva software (BD Biosciences).
2.5. Enzyme-linked immunosorbent assay (ELISA)
To determine the concentration of secreted TRAIL (sTRAIL), ASCs were cultured at diﬀerent seeding densities (5K–40K). After 5 days of culture, conditioned medium (CM) was recovered by centrifugation and then stored at −80 °C until analysis. Secreted TRAIL concentrations were measured using a human TRAIL Quantikine ELISA kit (R&D Systems) according to the manufacturer's instructions.
H460 cells were plated at 1 × 104 cells/cm2 in 96-well plates and cultured for 1 day. Since the CM of 40K-ASCs may be deficient in glucose and amino acids  and this nutritional deficiency may in-fluence H460 cell death, the CM obtained after a 5-day culture of 40K-ASCs was concentrated using centrifugal filter units (Millipore). The H460 cells were then treated with recombinant TRAIL (rTRAIL; R&D Systems) and concentrated- or normal CM from 40K-ASCs cultured for 1–5 days and further cultured for 1 day. Methylthiazolyldiphenyl-tet-razolium bromide (MTT; Sigma-Aldrich) dissolved in PBS was added to each well (final concentration: 5 mg/ml) and incubated at 37 °C for 2 h. MTT formazan was dissolved in 100 μl dimethyl sulfoxide and
incubated for a further 15 min with shaking before measuring the op-tical density at 570 nm of each well on a microplate reader (BioTek Instruments, Winooski, VT, USA).
The PE-Annexin-V apoptosis detection kit I (BD Biosciences) was used according to the manufacturer's instructions. Cells were harvested, washed twice with cold PBS, and re-suspended in binding buﬀer. Cells were stained with PE-Annexin-V and 7-aminoactinomycin D (7-AAD) for 15 min at room temperature in the dark. Cells were then analyzed without washing on a flow cytometer (BD FACSAria III) within 1 h.
2.8. Cell cycle analysis
The cellular DNA content of live cells was analyzed by Vybrant DyeCycle Ruby staining (Molecular Probes, Eugene, OR, USA) ac-cording to the manufacturer's instructions. Briefly, H460∗ cells were directly co-cultured with 40K-ASCs for the indicated time periods, trypsinized, and resuspended in 0.5 ml DMEM. Cells were incubated with 5 μM ruby stain at 37 °C for 20 min in the dark. DNA content was evaluated on a flow cytometer (BD FACSAria III), and then the cell cycle stage of H460∗ was analyzed.