Crp 8

mCRP was detected in cellular supernatants (previously centrifuged at 1,000 g for 10 min) by an ELISA assay following the protocol recently described by Zhang et al. [52 (link)]. For this purpose, mouse anti-human CRP mAb CRP-8 (Sigma-Aldrich, C1688) was immobilized as capture antibody at 1:1,000 in coating buffer (10 mM sodium carbonate/bicarbonate, pH 9.6) overnight at 4°C. After washing three times for 2 minutes each with TBS, non-specific binding sites were blocked with filtered 1% BSA-TBS for 1 hour at RT. Samples diluted 1:100 in blocking buffer were added into wells for 1 hour at RT. Then, washing step was repeated and samples were incubated with sheep anti-human CRP polyclonal antibody (1:2,000 in blocking buffer) (BindingSite), prior incubation with a HRP-labeled donkey anti-sheep IgG (1:10,000 in blocking buffer) (Abcam). Signaling was detected with VersaMax Microplate Reader and The OD value of each sample was calculated as OD₄₅₀–OD₅₇₀ nm.

Human monocytes derived macrophages (HMDM) plated on a round caver glass at 16-mm dish were fixed in the absolute methanol for 10 min. After rinsing with cold PBS (pH 7.4) cells were permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature. After blocking, antihuman-CRP antibody clone CRP-8 (Sigma-Aldrich, USA) and goat anti-human IL-6 (1:00, R & D Systems, Inc, USA) were respectively added and incubated at room temperature for 2 hours followed by incubation with CyTM3 conjugated Donkey Anti-mouse IgG (Jackson Immuno Research Labs, Baltimore, USA) and FITC conjugated Rabbit Anti-Goat IgG (Jackson Immuno Research Labs, Baltimore, USA) for 1 hour. Following removal of the antibodies, and nucleus counterstaining with To-pro-3 iodide (Invitrogen, Carlsbad, Ca, USA). Cells were rinsed with PBS, cover glass with cells removed from dish and mounted with UltraMount (Lab Vision, UK) [22 ]. All control samples were processed without primary antibody. Fluorescence was immediately observed using either an Axioscop 2 or Leica laser scanning confocal microscope (Bensheim, Germany) with image processing software “Image Pro Plus version 6”. Light intensity and contrast were standardized for respective cells samples with an appropriate control.

The general procedures for ELISA were as follows: All ELISA analysis was carried out on a sterile, 96-well plate (Costar). 50 μl of each sample was added to each well. Plates were blocked with 3% bovine serum albumin (BSA) and washed with phosphate buffered saline (PBS)-Tween (Sigma). The primary antibody used was a monoclonal anti-C-reactive protein antibody (CRP-8, Sigma) produced in a mouse; the secondary antibody was an anti-mouse IgG (whole molecule)–peroxidase antibody produced in rabbit (Sigma). Both were used at 1:40,000 dilution. Positive and negative controls were included in analysis and all samples were tested in triplicate. The developing substrate used was 3, 3′, 5, 5′, tetramethylbenzidine (TMB) liquid substrate (Sigma). ELISA plates were left to develop for 10 min prior to addition of 2 M sulfuric acid. Plates were read at 450 nm on a BioTek EL800 plate reader using Gen5 software.