IP-MS against LPS-stimulated PBMCs
Peripheral Blood Mononuclear Cells (PBMCs) were isolated from heparin-treated whole blood from a healthy donor by dilution of blood sample with equal volume of sterile PBS (Sigma-Aldrich) and adding blood cell density gradient media Ficoll-PAQUE (GE Healthcare). After centrifugation at 400 x g for 20 min with the brakes off, the layer of PBMCs on the top of Ficol-PAQUE solution was collected and washed with PBS. Viable cells were counted by staining with trypan blue (Sigma-Aldrich).
Immunoprecipitation was performed according to the method described earlier2. Peptide identification was performed using a Dionex Ultimate 3000 nano-LC system coupled via electrospray ion source (ThermoFisher Scientific) to a Q-Exactive Orbitrap (ThermoFisher Scientific). MS raw files were analyzed by MaxQuant software (Version 1.5.5.1) and searched against human Uniprot/Swissprot database (release-2018_04). Normalized Spectral Abundance Factor (NSAF) values were calculated as described in2.
IL6RA Dimerization Assay in collaboration with DiscoverX/Eurofins
Example Protocol: U2OS IL6RA/IL6ST Dimerization Assay (Anti-Ligand)
The U2OS IL6RA/IL6ST cell line was maintained in Culture Kit 103. Cells were maintained by splitting 1:3 every 2-3 days, when cells reached ~80% confluence. To evaluate responsiveness to anti-IL-6 binders, 20μl of cells were seeded at 5K/well in a 384 well plate in CP5 plating reagent and allowed to adhere for 24hr in a humidified incubator at 37 ºC/5% CO2.
A dilution series of each antibody (1:3 dilutions, 11pt-DRC) were prepared as 10X stocks in 0.1%BSA/PBS in triplicate for each dose. No antibody was added to the last well (e.g. lowest dose) in each DRC (to serve as vehicle only control). A 10X stock of the stimulation dose of IL-6 (1X dose= 3ng/mL) was prepared and mixed 1:1 with the antibody dilution series, then incubated for 90’ at room temperature (R/T). 5 μL of the antibody/ligand premix was added to the appropriate wells containing cells, then incubated in a humidified
incubator at 37 ºC/5% CO2 for 4.5 hrs.
25 μL of PathHunter FLASH Detection Reagent (prepared per package insert) was added to each well. Plates were incubated for 60 min at room temperature in the dark prior to reading luminsescence signal on an Envision luminometer. Data were plotted using GraphPad Prism 6; EC-50 values were calculated using a sigmoidal dose response curve fit with variable slope (four parameter) with no constraints; fit method= least squares (normal fit).
HepG2 STAT3 Reporter Assay in collaboration with DiscoverX/Eurofins
Example Protocol: HepG2 STAT3 Reporter Assay (Anti-Ligand)
The HepG2 STAT3 Reporter cell line, which endogenously expresses the IL-6 receptor, was maintained in Culture Kit 103. Cells were maintained by splitting 1:3 every 2-3 days, when cells reached ~80% confluence.
To evaluate responsiveness to anti-IL-6 binders, 20μl of cells were seeded at 5K/well in a 384 well plate in CP5 plating reagent and allowed to adhere for 24hr in a humidified incubator at 37°C / 5% CO2. A dilution series of each antibody (1:3 dilutions, 11pt-DRC) were prepared as 10X stocks in 0.1% BSA / PBS in triplicate for each dose. No antibody was added to the last well (e.g. lowest dose) in each DRC (to serve as vehicle only control). A 10X stock of the stimulation dose of IL-6 (1X dose= 0.3 ng/mL) was prepared and mixed 1:1 with the antibody dilution series, then incubated for the indicated time (90’) at room temperature (R/T).
5 μL of the antibody/ligand premix was added to the appropriate wells containing cells, then incubated in a humidified incubator at 37°C / 5% CO2 for 16 hr. 25 μL of PathHunter ED Detection Reagent (prepared per package insert) was added to each well. Plates were incubated for 60 min at room temperature in the dark prior to reading luminescence signal on an Envision luminometer. Data were plotted using GraphPad Prism 6; EC-50 values were calculated using a sigmoidal dose response curve fit with variable slope (four parameter) with no constraints; fit method= least squares (normal fit).