Sunrise photometer

OVA-specific serum IgG was measured by ELISA and normalized to a standard serum (arbitrary units, AU). 96-well microtiter plates (Nunc MaxiSorp^™, Affymetrix eBioscience, Santa Clara, CA, USA) were coated with 0.5 μg OVA per well (Sigma-Aldrich, St. Louis, MO, USA) in coating buffer (Candor Bioscience GmbH, Wangen, Germany) overnight at 4°C, washed with PBS/0.05% Tween20^™ and blocked with PBS/0.05% Tween20^™/10% FCS. IgG binding was detected using goat anti-mouse IgG coupled to HRP (Southern Biotech, Birmingham, AL, USA) and BD OptEIATM TMB Substrate Reagent Set (BD, Franklin Lakes, NJ, USA). Optical density at 450 nm was measured with the Tecan Sunrise photometer (Tecan Group Ltd., Maennedorf, Switzerland).
The relative affinity of TNP-specific IgG antibodies was determined based on the quantification of binding to differently haptenized bovine serum albumin (BSA)–TNP₂-BSA and TNP₂₆-BSA (adapted from [34 (link)])–as the carrier protein. IgG binding to TNP-BSA (Biosearch Technologies, Petaluma, CA, USA) was measured by ELISA as described above, and relative affinity was calculated as the ratio of the binding to TNP₂ to TNP₂₆.

After differentiation of 3T3-L1 fibroblasts to mature adipocytes, cells were fixed in 10% formaldehyde/PBS in two incubation steps, 5 min and 1 h, and subsequently washed with 60% isopropanol. Oil Red O (ORO) stock solution was prepared by solving 3.5 g/l ORO in isopropanol and stored at 4 °C. The working solution was prepared fresh before usage by diluting the stock solution in dH₂O 60:40, incubation at RT for 20 min, and filtering (mesh size: 0.2 µm) resulting in a final concentration of 2.1 g/l ORO. For staining, fixed cells were incubated with fresh working solution of ORO for 10 min. After incubation, cells were immediately washed four times with tap water. Pictures were taken using Leica LAS EZ software (Leica Microsystems GmbH, Wetzlar, Germany) and further analyzed using ImageJ, modifying an approach described before [48 (link)]. Elution of ORO was carried out by adding 50 µl isopropanol (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and short incubation at RT while pipetting up and down. OD values were measured at 500 nm using the Sunrise™ photometer (Tecan Group Ltd., Männedorf, Switzerland).

Cell culture density was measured using different systems. Proliferation of H1299 cells was monitored via the xCELLigence system (Roche). Because the xCELLigence systeme cannot be applied to suspension cells, a Vi-CELL XR cell counter (Beckman Coulter) was used to measure proliferation of Raji cells transfected with Brd4 constructs. The long-term impact of JQ1 on Raji cells was detected with a Countess automated cell counter (Invitrogen, Thermo Fisher Scientific).
Cell proliferation at increasing JQ1 concentrations was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay System (Promega). Cells were seeded in a 96-well plate and increasing concentrations of JQ1 or DMSO (control) were added. After 72 h MTS tetrazolium compound was added to each well for one hour. Then the quantity of the MTS formazan product was measured as absorbance at 490 nm with a Sunrise photometer (TECAN) which was operated using the Magellan data analysis software (v7.2, TECAN). Relative signals were calculated by dividing the JQ1 signals by the corresponding DMSO signals.