5 bromo 4 chloro 3 indolyl phosphate nitro blue tetrazolium

MSC were seeded at 600 cells per well in 384-well plate in MSC culture medium and treated as previously described. After 72 h, MSC medium was replaced by STEMPRO osteogenic induction medium (Invitrogen) in the presence or not of the different drugs. After 7 days of treatment, cells were fixed with ethanol 95% and stained by adding either a colorimetric substrate of the alkaline phosphatase, the 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma-Aldrich) or a chromogenic substrate of this enzyme (absorbance at 405 nm), the p-nitrophenyl phosphate (Pierce Biotechnology, Rockford, IL, USA).

Proteins from SDS gels were transferred to polyvinylidene difluoride membranes and immunoblotted with antibodies indicated in the figures, followed by incubation with the corresponding secondary antibodies (Sigma-Aldrich, St. Louis, MO). Signals were measured by incubating the blots with 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (Sigma-Aldrich). Blots were scanned with a photoscanner and quantified with Scion Image software. Antibodies used included monoclonal anti-EIF3h (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-actin (Sigma).

Multiscreen 96-well plates (Millipore) were coated with 50 μl sterile PBS (Tritium) containing either 5 μg/ml MenC-polysaccharide (NIBSC) conjugated to human serum albumin (NIBSC), 5 μg/ml tetanus toxoid (Baxter), 10 μg/ml goat-anti-human IgG (Cappel) or 10 μg/ml goat-anti-human IgA (Cappel) or solely PBS (blank wells). Coated plates were stored at 4°C until use.
After 5 days of polyclonal stimulation, B cells were harvested, washed, resuspended in AIMV medium (Gibco, Invitrogen) and counted. Wells were blocked with 100 μl AIMV medium for 1 hour before use and filled with 50 μl of cell suspension containing 1x10⁵ B cells. The plates were incubated ON at 37°C and 5% CO₂. The next day, wells were washed with PBS/0.05% Tween20 (Merck) and goat-anti-human IgG (Sigma Aldrich, 1/5000) or goat anti-human IgA (Sigma 1/2500) was added to bind to secreted IgG or IgA, respectively. After 1 hour of incubation at 37°C, the wells were washed and bound conjugate revealed by adding a substrate (5-bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium, Sigma). Spots were counted using an automatic ELISpot reader (C.T.L. Immunoscan). The number of MenC-PS-specific and TT-specific memory B cells per sample was measured in triplo and the mean number of these three measurements was used for the statistical analyses.

Dot blot assay was carried out with extracted LPS of various leptospiral serogroups. The optimal LPS concentration for IgM based detection was determined as 1.5 μg (S1 Fig). Antigens were dotted on the nitrocellulose membrane and air dried. The membrane was blocked with 4% non-fat milk and incubated at 37°C for 1 hour and washed four times with PBS-T. The membrane was incubated with patients’ sera diluted in PBS-T (1: 200) for 1 hour at 37°C. The membrane was incubated with anti-human ALP conjugated-IgM (1:2000; Sigma Aldrich, USA) at 37°C for 1 hour and washed with PBS-T. The membrane was further developed with chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate Nitro-blue tetrazolium (Sigma Aldrich, USA) and the intensity was quantified densitometrically and expressed as arbitrary units (AU).

Samples (3 μg of Act d 2 and dAct d 2) were separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and replica gels were electrotransferred onto polyvinylidene difluoride (PVDF) membranes. After blocking (Sigma-Aldrich, St. Louis, MO), membranes were incubated with a pool of patient sera (1:2 dilution in PBS) overnight, washed three times with PBS-Tween 20 0.05% for 10 min, then incubated with goat antihuman IgE-peroxidase conjugate (Invitrogen; 1:5000 dilution in PBS). IgE-binding components were detected by means of chemiluminescence (Amersham Biosciences, Little Chalfont, UK). Alternatively, blocked membranes were immunodetected with rabbit polyclonal antibodies to peach TLP (1:10.000 dilution; 1 h; kindly provided by Dr. C. Pastor, FJD, Madrid, Spain), with anti-complex asparagine-linked glycan serum (Faye et al. 1993 (link)) (1:10.000 dilution; 1 h) or with anti-xylose and anti-fucose antibodies (also kindly provided by Dr. C. Pastor, FJD), and then treated with anti-rabbit IgG-phosphatase alkaline conjugate (Sigma; 1:5000 dilution) and developed with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma).

Cellular immunity was assessed by a T-cell stimulation assay. PepMix™ SARS-CoV-2 peptide pools were purchased from JPT (Berlin, Germany). The pools comprised 15-mer peptides overlapping by 11 amino acids (aa) covering the entire sequences of the SARS-CoV-2 spike protein. The spike peptides were split into two sub-pools, S1 (aa 1-643) and S2 (aa 633-1273). Peptides were dissolved in dimethyl sulfoxide and diluted in an AIM-V medium for use in the ELISpot assays. For ex vivo ELISpot assays, PBMCs were thawed. A total of 1–2 × 10⁵ cells per well were incubated with SARS-CoV-2 peptides (2 μg/mL; duplicates), AIM-V medium (negative control; 3–4 wells), or PHA (L4144, Sigma, St. Louis, MO, USA; 0.5 μg/mL; positive control) in 96-well plates coated with 1.5 μg anti-IFN-γ (1-D1K, Mabtech, Stockholm, Sweden) for 24 h. After washing, spots were developed with 0.1 μg biotin-conjugated anti-IFN-γ (7-B6-1, Mabtech), streptavidin-coupled alkaline phosphatase (Mabtech, 1:1000), and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma). Spots were counted using a Bio-Sys Bioreader 5000 Pro-S/BR177 (Karben, Germany) and Bioreader software generation 10. T-cell responses were considered positive when mean spot counts were at least threefold higher than the mean spot counts of the unstimulated wells.

For IFN-γ enzyme-linked immunospot assay, nitrocellulose-bottomed 96-well plates (MultiScreen HTS-IP, Millipore) were coated overnight at 4°C with anti-IFN-γ mAb (AN18 Mabtech). Plates were blocked with 10% FBS-containing medium for at least 30 min at room temperature. Erythrocyte-depleted spleen cells from immunized or naive mice were plated in triplicates at four different cell concentrations (1×10⁶ to 1.25×10⁵ c/well) including HER-2⁺ cell line lysate as antigen (5:1 responder to stimulator ratio). Plates were then incubated overnight at 37°C, washed five times and incubated with biotinylated anti-IFN-γmAb (R4-6A2-biotinMabtech) for 2 hours at room temperature. After washing, streptavidin-enzyme conjugate (Mabtech) was added to each well and incubated for 1 hour at room temperature, followed by 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma). The reaction was terminated on the appearance of dark spots by washing the plates with tap water. The spots were counted using the 4-Plate ELISPOT Reader V2.1 (Aelvis GmbH).