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Customer kindly contacted us regarding human PBC IL8 production stimulation by ab50086. Specifically asking for references / data indicating this stimulation.
Asked on Dec 14 2012
I contacted our laboratory that used ab50086 to induce IL-8 production and they provided the following response:
"With reference to your query, the detailed protocol for the dose-dependent stimulation of IL-8 production by human PBMC which we employ to measure biological activity of ab50086 is as follows:
Human 4-IBB Ligand (4-IBBL) Stimulation of PBMC and Measurement of Induced Cytokines including TNF-a and IL-8
Stimulation of human PBMC by 4-1BBL is best observed using freshly isolated human PBMC. You can use RPMI alone or with added FCS or heat-inactivated human AB serum (1%) v/v. First add cells to the wells (0.5 mL of 5 X 106 /mL). Then soon thereafter, add (0.5 mL) increasing concentrations of 4-1BBL (l to 100 ng/mL, final concentration) to the cells. Place the plate in the incubator for 24 hours. Remove the supernatant carefully so that you do not gather cells. Alternatively, aspirate the well contents and centrifuge at 450 g for 15 minutes and measure IL-8 in the supernates. Some donors do not respond and the reason is unclear. Extending the time of incubation to 48 can also be tested but again it is donor variable.
Do not add 4-1BBL to the plastic well but only to the cells already in the Wells.
Measurement of other cytokines, including TNF-a is also possible but IL-8 is a reliable cytokine. Induction of cytokines should always include two controls: a negative control (no 4-1BBL) and a positive control. We recommend as the positive control adding 10 ng/mL of E. coli endotoxin (Sigma). If you culture the PBMC with 1% human AB serum, the negative control (no stimulant) will be higher for IL-8 production.
This assay can be done in a 96 flat bottom well plate with a maximum of 200 mL per well. Add 100 mL of cells then 100 mL of 4-1BBL. Again, the final concentration will be 1 to l00ng/mL in the wells. We have found that donor variability depends on concentration. For example, 1, 10, 20, 40 and 80 ng/mL final concentration would be the best dose-response to detecting a response.
The ED50 for this effect is 5-10 ng/ml. Please note results may vary with PBMC donors"
Answered on Dec 14 2012