Bcip nbt

Manufactured by Mabtech

Sourced in Germany

BCIP/NBT is a chromogenic substrate used for the detection and visualization of alkaline phosphatase activity in various biochemical and immunohistochemical applications. It produces a dark blue-purple precipitate at the site of the enzyme reaction.

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Lab products found in correlation

9 protocols using bcip nbt

SARS-CoV-2 Spike IgG ELISpot Assay

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SARS-CoV-2 and total IgG ELISpot assays were performed using Mabtech IgG ELISpot kits (Mabtech; Cincinnati, OH). Cryopreserved PBMCs (T4 and T7 collection timepoints) were thawed, counted, and resuspended in complete medium RPMI 1640 (Corning; Corning, NY) supplemented with 10% fetal bovine serum (Fisher Scientific; Waltham, MA). After resting the cells for 30 min at 37°C, 3 × 10⁵ cells were stimulated with 1 μg/mL R848 and 10 ng/mL rhIL-2 to induce IgG secretion by memory B cells and then incubated at 37°C, 5% CO2 for 3 days. PVDF plates were coated with mAbs91/145 one day before performing ELISpot. 1 × 10⁵ and 1 × 10⁴ stimulated cells were seeded in coated plates for SARS-CoV-2 spike-specific IgG and total IgG detection, respectively, and the cells were incubated at 37°C, 5% CO2 for 24 h. RBD-WASP and mAbs MT78/145-biotin were used to detect spike-specific IgG and total IgG, respectively. Detection solution containing anti-WASP-ALP and streptavidin-HRP was then added to the cells and incubated at room temperature for 1 h. BCIP/NBT (Mabtech, Cincinnati, OH) and AEC substrates (BD Biosciences; San Diego, CA) were then used to reveal the presence of spots. Spots formed by IgG-secreting cells were imaged by dissection microscopy and counted using ImageJ.

Huang Y., Shin J.E., Xu A.M., Yao C., Joung S., Wu M., Zhang R., Shin B., Foley J., Mahov S.B., Modes M.E., Ebinger J.E., Driver M., Braun J.G., Jefferies C.A., Parimon T., Hayes C., Sobhani K., Merchant A., Gharib S.A., Jordan S.C., Cheng S., Goodridge H.S, & Chen P. (2022). Evidence of premature lymphocyte aging in people with low anti-spike antibody levels after BNT162b2 vaccination. iScience, 25(10), 105209.

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Quantifying Interferon-alpha Secretion in pDCs

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ELISpot assays were conducted using the Human IFN-α2 ELISpot BASIC (ALP) kit (Mabtech) following manufacturer’s instructions. Before seeding of cells, plates were pre-coated with coating antibody. Sorted pDCs were added to wells containing 2.5x10⁵ non-sorted PBMCs at 10–500 pDCs per well. 1000 non-pDCs (lymphocyte, singlet, live cells, CD3-CD20-, HLA-DR+, CD14-, CD123-) were added into a separate set of wells. Cells were incubated with 2.5 μg/mL of the TLR7/8 agonist, CL097 (InvivoGen) and incubated for 48 hours at 37°C. The plate was incubated with the biotinylated detection antibody for 2 hours at room temperature followed by incubation with Streptavidin-ALP for 1 hour at room temperature. BCIP/NBT (Mabtech) was then added to the plate, which was allowed to develop for 15 minutes. Plates were scanned using the Immunospot Analyzer (Cellular Technology Limited) and spots counted with ImageJ.

Lee M.Y., Upadhyay A.A., Walum H., Chan C.N., Dawoud R.A., Grech C., Harper J.L., Karunakaran K.A., Nelson S.A., Mahar E.A., Goss K.L., Carnathan D.G., Cervasi B., Gill K., Tharp G.K., Wonderlich E.R., Velu V., Barratt-Boyes S.M., Paiardini M., Silvestri G., Estes J.D, & Bosinger S.E. (2021). Tissue-specific transcriptional profiling of plasmacytoid dendritic cells reveals a hyperactivated state in chronic SIV infection. PLoS Pathogens, 17(6), e1009674.

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Tumor-Specific T Cell Reactivity Assay

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Enzyme-linked immune absorbent spot (ELISPOT) assay was performed to assess TIL and PBMC reactivity against autologous tumor cell line (TCL) and selected neo-peptides using 96-well plates (Merck Millipore) coated with IFNγ capturing antibody (clone 1-D1K (Mabtech). TILs or PBMCs were co-cultured with autologous tumor cell lines or neo-peptides and incubated overnight at 37°C in 5% CO₂. The experiments were performed both with and without peptide pulsing. Following incubation, plates were washed, and biotinylated secondary antibody (clone 7-B6-1-Biotin, Mabtech) was added. Afterwards, plates were washed again, and Streptavidin-ALP (3010–10, Mabtech) was added. Finally, BCIP/NBT (3650–10, Mabtech) was added and catalyzed by the enzyme conjugate to form spots visualizing IFNγ release of a cell at that location. Spots were counted using an Immunospot 2.0 Analyzer (Cellular Technologies Limited).

Nielsen M., Monberg T., Sundvold V., Albieri B., Hovgaard D., Petersen M.M., Krarup-Hansen A., Met Ö., Camilio K., Clancy T., Stratford R., Sveinbjornsson B., Rekdal Ø., Junker N, & Svane I.M. (2023). LTX-315 and adoptive cell therapy using tumor-infiltrating lymphocytes generate tumor specific T cells in patients with metastatic soft tissue sarcoma. Oncoimmunology, 13(1), 2290900.

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IgG Monoclonal Antibody Detection

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Anti-human IgG monocloning antibodies (MT91/145, Mabtech) were diluted with PBS (PH7.4) and coated onto a 96-well polyvinylidene fluoride plate (Millipore) treated with 70% ethanol for 2 min at a concentration of 15 µg/mL (100 µL/well) overnight at 4°C. The plates were washed with PBS for five times (300 µL/well) and blocked using 10% bovine serum (200 µL/well) at room temperature for 1 hour. MB cells were pre-stimulated with a mixture of R848 at 1 µg/mL and rhIL-2 at 10 ng/mL (Mabtech) for 24 hours. Activated MB cells and CD8⁺ T cells were inoculated into the plates at a ratio of 1:4 and cultured at 37°C and 5% CO₂ for 48 hours. After washing the plate five times with PBS (300 µL/well), 100 µL (1 µg/mL) of biotin-anti human IgG mAb (MT78/145, Mabtech) was added to the plates and incubated at RT for 2 hours. After another round of washing, 100 µL streptavidin-Alkaline phosphatase (1:1000, Mabtech) was added and incubated for 1 hour at RT. 100 µL substrate solution (BCIP/NBT, Mabtech) were added and developed until the distinct spots emerge. The plates were analysed using a CTL reader (ImmunoSpot, Cleveland, Ohio, USA).

Wang K., Zhao J., Feng X., He S., Li J., Sun F., Xu Z., Yang H., Ye J., Cao L, & Ye S. (2024). PD-1/PD-L1 governed cross-talk of exhausted CD8+ T and memory B cells in systemic lupus erythematosus. RMD Open, 10(1), e003503.

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IFNγ ELISPOT Assay for T Cell Response

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IFNγ ELISPOT kits, including pre-coated plates and alkaline phosphatase (ALP), were bought from Mabtech. The assay was conducted following their protocols. ELISPOT assay strip plate was washed with PBS and blocked with RPMI 1640 containing 10% FBS. T cells were co-cultured with peptide only or T2 cells pre-pulsed with or without peptides (10 μg/ml) in the above pre-treated ELISPOT plate for 24 hours. The plate was rinsed with PBS, then added with alkaline phosphatase (ALP) labeled anti-human IFNγ mAb (7-B6-1-ALP, 1:200; Mabtech) for 2 hours. After rinsing, 5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium (BCIP/NBT, Mabtech) was used to develop the immune-spot. Spots were imaged and counted by an ELISPOT Reader (BioReader 4000, BIOSYS).

Li D., Chen C., Li J., Yue J., Ding Y., Wang H., Liang Z., Zhang L., Qiu S., Liu G., Gao Y., Huang Y., Li D., Zhang R., Liu W., Wen X., Li B., Zhang X., Zhang X, & Xu R.H. (2023). A pilot study of lymphodepletion intensity for peripheral blood mononuclear cell-derived neoantigen-specific CD8 + T cell therapy in patients with advanced solid tumors. Nature Communications, 14, 3447.

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IFN-γ Enzyme-Linked Immunospot Assay

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IFN-γ Enzyme-Linked Immunospot plates (MAHAS45, Millipore, Molsheim, France) were coated with anti-IFNγ antibody (eBiosciences, San Diego, CA, USA) overnight at +4°C. Stimulation media (complete RPMI), UTY (2 µg/mL), DBY (2 µg/mL), UTY + DBY (2 µg/mL), or Concanavalin A (Sigma, Lyon, France) (5 µg/mL) were plated and 5.10⁵ splenocytes/well were added. After 24 h of culture at +37°C, plates were washed and the secretion of IFNγ was revealed with a biotinylated anti-IFNγ antibody (eBiosciences), Streptavidin-Alcalin Phosphatase (Roche Diagnostics, Mannheim, Germany), and BCIP/NBT (Mabtech, Les Ulis, France). Spots were counted with an AID ELISpot Reader system ILF05 and the AID ELISpot Reader v6.0 software.

Vendomèle J., Dehmani S., Khebizi Q., Galy A, & Fisson S. (2018). Subretinal Injection of HY Peptides Induces Systemic Antigen-Specific Inhibition of Effector CD4+ and CD8+ T-Cell Responses. Frontiers in Immunology, 9, 504.

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Quantifying Antibody-Secreting B Cells

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Antibody-secreting B cells (plasma cells) were assessed by B cell ELISPOT assay according to the manufacture’s protocol (MabTech, Nacka Strand, Sweden). Briefly, ELISPOT plates were coated with capture anti-IgG or anti-IgM antibody (BD Pharmingen), and then spleen and bone marrow cell suspensions in 5% FBS/complete RPMI were added to each well and incubated for 24 h at 37°C in 5% CO₂. Plates were extensively washed to remove the cells and biotinylated anti-IgG or anti-IgM antibody was added followed by Streptavidin-ALP and substrate (BCIP/NBT; MabTech). After color development, total spots per well were quantified using an ImmunoSpot Series 2 Analyzer (Cellular Technology Ltd., Shaker Heights, OH). Priming of donor-reactive T cells to IFN-γ–producing cells was quantified by ELISPOT assay as previously described (19 (link), 20 (link)).

Abe T., Ishii D., Gorbacheva V., Kohei N., Tsuda H., Tanaka T., Dvorina N., Nonomura N., Takahara S., Valujskikh A., Baldwin WM I.I.I, & Fairchild R.L. (2015). Anti-huCD20 Antibody Therapy for Antibody-Mediated Rejection of Renal Allografts in a Mouse Model. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 15(5), 1192-1204.

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AAV Capsid-specific Cellular Immunity Assay

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Anti-AAV2 and anti-AAV8 cellular immune responses were evaluated with an IFNγ ELISpot assay using overlapping peptide libraries covering the whole AAV2 or AAV8 capsid sequences (15 per 10 mers, PEPscreen, Sigma) split into three peptide pools each. MultiScreenHTS filter plates, with polyvinyldiene difluoride membrane (PVDF, Millipore) were coated overnight at 4°C with human anti-IFNγ antibody (clone MT126L, Mabtech). After coating, 2 × 10⁵ cells per well were plated and restimulated 48H with AAV2- or AAV8- derived peptide pools at a final concentration of 10 μg/mL. Medium alone served as negative control, while cells stimulated with concanavalin A (Con A, 10 μg/mL, SIGMA) served as a positive control. After incubation with a biotinylated anti-IFNγ antibody (clone 7-B6-1, MabTech) and Streptavidin-ALP (MabTech), enzymatic reaction was revealed using NBT/BCIP (MabTech). Spot number was determined using an ELISpot iSpot Spectrum reader (AID, Strassberg, Germany) and analyzed with AID ELISpot reader Software V7.0 (AID, Germany). Responses were considered positive when the number of spot-forming colonies per million cells was >50 and at least threefold higher than the medium alone negative control. For positive responses, statistical analyses were performed using a DFR(2×) test (Distribution Free Resampling).

Vandamme C., Xicluna R., Hesnard L., Devaux M., Jaulin N., Guilbaud M., Le Duff J., Couzinié C., Moullier P., Saulquin X, & Adjali O. (2020). Tetramer-Based Enrichment of Preexisting Anti-AAV8 CD8+ T Cells in Human Donors Allows the Detection of a TEMRA Subpopulation. Frontiers in Immunology, 10, 3110.

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T-cell Response Assessment via IFNγ-ELISPOT

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T-cell responses toward IO103 and IO120 were assessed using IFNγ-ELISPOT, which measures the release of IFNγ from specific T-cells upon stimulation with the peptides. We followed the procedure described earlier.^{14 (link),17 (link)} To improve sensitivity, we stimulated the peripheral blood mononuclear cells (PBMC) in vitro once.^{24 (link)} We used 96-well PVDF plates (MultiScreen, MAIP N45; Merck Millipore, Burlington, MA) coated with anti-IFNγ-mAb (Mabtech, Nacka Strand, Sweden). Secondary biotinylated anti-INFγ-mAB, Streptavidin–enzyme conjugate and the enzyme substrate NBT/BCIP from Mabtech were used. Spots were counted using an ImmunoSpot Series 2.0 Analyzer (Cellular Technology Limited, Cleveland, OH), with an upper threshold of 500 spots/well. ELISPOT assays of PBMCs were done in triplicate with 2.5–3.0 × 10⁵ cells/well. ELISPOT assays of SKILs were done with varying numbers of cells and primarily done in triplicate, with results in duplicates highlighted in figures.

Klausen U., Grønne Dahlager Jørgensen N., Grauslund J.H., Munir Ahmad S., Gang A.O., Martinenaite E., Weis-Banke S.E., Breinholt M.F., Novotny G.W., Kjeldsen J.W., Orebo Holmström M., Pedersen L.B., Poulsen C.B., Hansen P.B., Met Ö., Svane I.M., Niemann C.U., Pedersen L.M, & Andersen M.H. (2021). An immunogenic first-in-human immune modulatory vaccine with PD-L1 and PD-L2 peptides is feasible and shows early signs of efficacy in follicular lymphoma. Oncoimmunology, 10(1), 1975889.

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