Cytokine human membrane antibody array kit

Cells were pretreated with or without 3-MA for 30 min and then incubated with cytokines for another 24 h. The conditioned medium (CM) was collected and centrifuged at 1,500 g for 5 min to remove cell debris. The supernatant was collected and stored at −20 °C for no more than 1 week. For cytokine array, CM was analyzed with Cytokine Human Membrane Antibody Array Kit (Abcam) following the manufacturer’s instruction. In brief, the array membranes were blocked by blocking buffer for 30 min and then incubated with CM overnight at 4 °C. After incubation with CM, membranes were incubated with biotin-conjugated anti-cytokines overnight at 4 °C and then with HRP-Conjugated Streptavidin for 2 h. The signal was detected with Detection Buffer and exposed on X-ray films (Kodak). For ELISA, CM was coated on a 96-well plate in 0.1 mol/L NaHCO₃ at 4 °C overnight. The coated plate was blocked with 5% BSA and then incubated with rabbit polyclonal anti-IP10 (Abcam) at 4 °C overnight. After incubation with the secondary antibody (1:500), tetramethylbenzidine (Thermo Scientific) was added for the color reaction. The reaction was stopped with 0.18 mmol/L H₂SO₄ and the absorbance was analyzed at 450 nm with a Synergy H1 Hybrid Reader (BioTek).

Cytokines secreted from human monocytic cells were determined using hybridizing medium with antibody-coated membranes (Cytokine Human Membrane Antibody Array Kit, Abcam, Cambridge, MA) according to the instructions from the manufacturer. Briefly, cells were added to serum-free medium in six-well plates and stimulated with CPSIT_P7 and cultivated overnight. The culture supernatants were collected, pooled (n = 3/group), and screened for inflammatory cytokines, and then mixed with the array membrane. A biotin-conjugated secondary antibody was used to detect cytokines by HRP-conjugated streptavidin. Signals were detected with an enhanced chemiluminescence system (Syngene, Cambridge, United Kingdom).

Construction of expression vector and transfections were performed as previously described [18 ]. Cytokine analysis using cell-cultured media and whole-cell extracts from A549 cells was performed using a human cytokine antibody array membrane kit (Abcam, Cambridge, UK) according to the manufacturer's protocols.