5 bromo 4 chloro 3 indolyl phosphate bcip nitro blue tetrazolium substrate solution

ESAT-6 and CFP-10 antigens were provided by Lionex Gmbh (Braunschweig, Germany, www.lionex.de).
Mouse monoclonal antibody (mAb) against human complement factor C3 and the split protein C3b (C3-C3b) was from Abcam, (Cambridge, UK) and polyclonal goat anti-mouse IgG antibodies (Abs) conjugated with alkaline phosphatase (AP) were from Dako (Glostrup, Denmark).
The 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitro blue tetrazolium (NBT) substrate solution, 2′,7′-dichlorodihydrofluorescindiacetate (H₂DCFDA), anti-mouse IgA, anti-mouse IgG, and o-phenylenediamine dihydrochloride (OPD) substrate were purchased from Sigma-Aldrich Co. and molecular weight marker was from Thermo Fisher Scientific, Spain.
Cell Titer 96^® AQueous One Solution Cell Proliferation Assay kit, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and endotoxin test were from Promega Biotech Ibérica, (Madrid, Spain) and Associates of Cape Cod, Inc. (Falmouth, MA, USA), respectively.
Phorbol 12-myristate-13-acetate (PMA) was from Abcam (Cambrige, UK), lipopolysaccharide (LPS) from InvivoGen (Toulouse, France), and phytohaemagglutinin (PHA) from Sigma-Aldrich Co.
The MILLIPLEX^® MAP Kit used for cytokine profile characterization was provided by Millipore, Merck KGaA (Darmstadt, Germany).

H1N1-virus-specific antibody-secreting cells (ASCs) in the spleens of H1N1-challenged WT and db/db mice were identified by ELISPOT^{64 (link)}. Briefly, 96-well plates (Cat. # MSIPS4W10, Millipore) were pre-coated with purified H1N1 influenza virus A/PR/8/34 (5 mg/mL) at 4 °C overnight. Plates were washed and blocked with RPMI 1640 with 10% FBS at room temperature for 1 h. 1 × 10⁴ PI^-CD3^-B220⁺ cells from spleens of WT and db/db mice with H1N1 influenza virus on 9 days post-challenge were sorted and cultured for 12 h. Goat Anti-Mouse IgG Alkaline Phosphatase was then added after thoroughly wash and incubated for 1 h at room temperature. 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP)/Nitro Blue Tetrazolium (NBT) substrate solution (Sigma–Aldrich) was added. Spots were monitored, stopped by gently washing, identified and counted by the Photoshop CS4 software (Adobe).