Human l507 array slides

Cytokines in culture media were detected and analysed using Human L507 Array slides (AAH-BLG-1–2, RayBiotech, Norcross, GA) by H-Wayen Biotechnologies (Shanghai, China). In brief, media without FBS was harvested from SKOV3/Vector and SKOV3/Wip1 cells after incubation for 48 h. In general, the concentrations of CMs were measured with a BCA protein assay kit (ThermoFisher Scientific Inc., USA) using a VICTOR Nivo multimode plate reader (PerkinElmer, USA). The Human L507 Array slides were dried for 2 h at room temperature and incubated with 800 μL blocking solution at room temperature for 30 min. After decanting the blocking buffer from each subarray, 400 μL diluted culture media was added and incubated overnight at 4 ℃. After decanting the samples, all arrays were washed 3 times with 800 μL of 1 × wash buffer I. Then, the slides were incubated with Cy3-conjugated streptavidin solution and rinsed with 1 × wash buffers I and II. The results were assessed using a GenePix 4000 B Scanner (Axon Instrument, USA) and analysed with the GenePix Pro 6.0 program (Axon Instrument, USA). Global normalization of the fluorescent spots was conducted to reduce noise and variation among the samples.

CMs were harvested from BEAS-2B, primary AECs and hiPSC-AEC cultures for secretome anaylsis. The concentration of CMs was measured with BCA protein assay kit (Pierce, Rockford, I11) using Multi-Skan FC (Thermo, USA). The purities of purified CMs were confirmed on UV spectrum. The Human L507 Array slides (RayBiotech, Norcross, GA) were dried for 2 hr at room temperature and incubated 400 μl of blocking solution at room temperature for 30 min. After decanting the blocking buffer from each sub array, 400 μl of diluted CM was added and incubated for 2 hr at room temperature. After decanting the samples, all arrays were washed 3 times with 800 μl of 1X wash buffer I and incubated with biotin-conjugated anti-cytokine antibodies for 2 hrs. Then, the slides were incubated with Cy3-conjugated streptavidin solution and rinsed with de-ionized water. The result was scanned using GenePix 4100 A Scanner (Axon Instrument, USA) and analyzed with GenePix Pro 7.0 program (Axon Instrument, USA). Global normalization was conducted in the fluorescent spots to reduce noise and variation among the samples.