Human cytokine chemokine array kits

BMFs (1 ×105) and MKN28 cells (1 ×105) were cultured alone or together in 2 ml of RMPI 1640 complete medium in a 6-well plate. Twenty-four hours later, cells were washed twice with PBS and incubated with fresh serum-free RMPI 1640 for another 24 hours. Then, the BMF-conditioned medium (BMF-CM), MKN28-CM and Co-Culture-CM were collected for antibody array. The mouse cytokine/chemokine array kits (Ray Biotech Inc. Atlanta, GA) were used to detect a panel of 24 secreted cytokines and chemokines in BMF-CM and Co-Culture-CM. The human cytokine/chemokine array kits (Ray Biotech Inc.) were used to detect a panel of 24 secreted cytokines and chemokines in MKN28-CM and Co-Culture-CM according to the manufacturer’s recommended protocol. The spot signal densities of array membrane were scanned using the Odyssey Infrared Imaging System (Li-Cor Biosciences). The raw numerical densitometry data were extracted from background. The data of samples were normalized to the positive control signals. The signal intensity for each antigen-specific antibody spot was proportional to the relative concentration of the antigen in that sample. The experiments were repreated twice (n=3).