Topro 3 or dapi

FITC-conjugated monoclonal antibodies (mAbs) to CD14, CD19, CD20, and CD66b; PE-conjugated mAbs to CD19, CD20, CD54, and CD105; PC7-conjugated mAbs to CD19, and CD20; and PB-conjugated mAbs to CD15 were provided by Beckman Coulter. Alexa-647-cojugated mAb to CD105 was provided by ebioscience. Appropriate isotype-matched mAbs were used as negative controls and all analyses were performed using a Gallios (Beckman Coulter) flow cytometer. Cell death was checked using TOPRO-3 or DAPI (Life Technologies) staining. Apoptosis evaluation was performed using PE-conjugated active caspase-3 apoptosis kit (Becton Dickinson).

Cells grown on cover slips were treated with OH-ME as indicated. DMSO served as solvent control, while N-OH-PhIP was included as positive control. Briefly, cells were fixed in ice-cold methanol at −20 °C for γH2AX staining or 4% paraformaldehyde (PFA) at room temperature for cytochrome c staining followed by blocking in PBS containing 5% (w/v) bovine serum albumin (BSA) and 0.3% (v/v) Triton X-100 for 1 h. Sample preparation for immunofluorescence staining of activated Bax included a fixation with 4% PFA followed by permeabilization with 0.2% CHAPS in PBS and a blocking step with PBS containing 5% (w/v) BSA. Immunofluorescence staining was essentially conducted as described [52 (link)] using the primary and secondary Alexa Fluor 488-conjugated antibodies listed in supporting Table S3. DNA was counterstained with TO-PRO-3 or DAPI (Life Technologies, Darmstadt, Germany) for 15 min. Finally, cells were mounted using VectaShield® (Vector Labs, Burlingame, CA) and analyzed by confocal microscopy using a Zeiss Axio Observer 7 microscope equipped with a 63x oil objective (Plan-Apochromat 63x/1.40 DIC M27) and a LSM900 confocal laser scanner (Zeiss, Oberkochen, Germany). Images were acquired by ZEN software version 3.4 (Zeiss) and processed using ImageJ software (NIH, MD).