F ab 2 goat anti rabbit igg h l alexa fluor 488

To quantify the NQO1 protein content in circulating monocytes, in-cell Western assays were performed as recently described by our group [15 (link), 16 (link)]. For that purpose, monocytes were fixed with formaldehyde and permeabilized using Triton X-100 in 96-well plates. Cells were blocked using 1% bovine serum albumin overnight at 4°C, then incubated with the primary antibodies rabbit anti-human NQO1 (ab34173, Abcam, UK) or rabbit anti-human ACTB (sc-130656, Santa Cruz Biotechnology, Germany). After washing, cells were incubated with the fluorescence-labeled secondary antibody F(ab′)₂-goat anti-rabbit IgG (H+L) Alexa Fluor 488 (Fisher Scientific, Denmark). All measurements were performed in triplicate, and the NQO1 protein content was analyzed relative to the ACTB protein content as an internal reference. Imaging was performed using an EnSpire Multimode Plate Reader (PerkinElmer, Denmark) at 520 nm emission with an excitation wavelength of 490 nm.

Protein was extracted from monocytes using an extraction reagent for mammalian cells including a protease inhibitor cocktail (cOmplete Lysis-M, pH 7.6, Roche, Denmark). Proteins were separated by 10% sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (10% RunBlue SDS gel, Expedeon, UK) at 120 V for 45 minutes and transferred to polyvinylidendifluoride membranes at 100 V for 75 minutes (Immun-Blot LF PVDF, Bio-Rad, USA). Membranes were blocked with blocking buffer (Superblock, Thermo Fisher Scientific, USA) for 1 hour at room temperature and incubated with the primary antibodies rabbit anti-human NQO1 (ab34173, Abcam, UK) or rabbit anti-human ACTB (sc-130656, Santa Cruz Biotechnology, Germany). After washing with tris(hydroxymethyl)aminomethane-buffered saline, the membranes were incubated with the fluorescence-labeled secondary antibody F(ab′)₂-goat anti-rabbit IgG (H+L) Alexa Fluor 488 (Fisher Scientific, Denmark). Imaging was performed using a Carestream Imager 4000pro (Fisher Scientific, Denmark) at 535 nm emission with an excitation wavelength of 470 nm.