Cytokine Array – Human Cytokine Antibody Array (Membrane, 23 Targets) (ab133996)

Human peripheral blood cells (1x10⁶ cells/mL) were cultured in RPMI media supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate.

Cells were cultured unstimulated or stimulated with 10 µg/mL PHA. Conditioned media was harvested after 48 hours, aliquoted and assayed using ab133996. Media alone was used as a negative control.

Cells were cultured unstimulated or stimulated with 10 µg/mL PHA. Conditioned media was harvested after 48 hours, aliquoted and assayed using ab133996. Media alone was used as a negative control. Samples were incubated on the human cytokin antibody arrays and results were quantified using the ULTRAQuant software.

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The human cytokine antibody array containing 23 target proteins was used to determine the effect of lipopolysaccharide (LPS) on the induction of cytokine production in macrophages. One million macrophage cells were plated in two sets and used in the study, one set was treated with PBS alone while the other set is treated with LPS at 1 µg/mL for 24 h. After 24 h of LPS treatment, both sets of macrophages were harvested using the provided cell lysis buffer and the cells were mechanically disrupted using a 20-gauge needle. Disrupted cell lysates were centrifuged and the supernatant was collected. The protein concentration of the supernatant was determined and 200 µg of proteins diluted in the blocking buffer was applied into previously blocked membranes and stored overnight at 4 °C. The remainder of the experiment was performed using the manufacturer’s instructions where all remaining incubations were performed at room temperature for 2 h. After incubation and washing steps, the proteins were detected using the provided detection reagents and photographed in a blot imaging system. Membranes were exposed for 15 s.