Patient peripheral blood mononuclear cells (PBMC) were isolated by ficoll-paque density gradient centrifugation. A stimulation cocktail containing 1 µg/mL R848 (Mabtech, Nacka Strand, Sweden) and 10 µg/mL IL-2 (Mabtech) was added for 72 h at 37°C/5% CO2 to activate antibody production by memory B cells. The frequency of total IgM and IgG-producing cells in PBMC was determined using IgM or IgG ELISpot assays. Briefly, anti-IgM or IgG capture antibodies (Mabtech) were incubated in high protein-binding PVDF membrane plates (Millipore, Billerica, MA) for 5 h at RT. Activated cell suspensions were seeded in duplicate at 5,000, 2,500 and 1,250 cells/well and incubated for 8 h at 37°C/5% CO2. Detection was performed by incubating biotinylated anti-IgM (Mabtech) or anti-IgG (Mabtech) detection antibodies for 2 h at RT. Streptavadin-conjugated alkaline phosphatase (R&D, Minneapolis, MN) was added for 2 h at RT followed by incubation with BCIP/NBT substrate (R&D) for 30 min at RT. The reaction was stopped by rinsing wells with ultrapure water. In parallel experiments to assess MDA-specific antibody-secreting cells, ELISpot plates were coated with 62.5 µg/ml MDA-modified BSA for 5 h RT. Cell suspensions were added in duplicate at 50,000, 25,000 and 12,500 cells/well and incubated for 8 h at 37°C/5% CO2. ELISpot detection was performed as for total IgM and IgG detection.
Bcip nbt substrate
Manufactured by R&D Systems
Sourced in Mongolia, United Kingdom
BCIP/NBT substrate is a chromogenic substrate solution used in immunological and histochemical applications for the detection and visualization of target proteins or enzymes. The substrate is composed of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT), which undergo a color reaction upon enzymatic cleavage, resulting in a blue-purple precipitate at the site of the target analyte.
Automatically generated - may contain errors
3 protocols using bcip nbt substrate
1
Quantification of IgM and IgG Antibody-Secreting Cells
2
ELISPOT Assay for Cytokine Measurement
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T-cell responses were measured essentially as described [14 (link),19 (link)]. Briefly, ELISPOT plates were coated with capture antibody specific for the cytokine and species of interest (mouse, monkey, or rabbit IFNγ or IL-5; all obtained from commercial sources including MABTECH AB [Cincinnati, OH, USA] and Cell Sciences [Newbury Port, MA, USA]), incubated, washed, and blocked. Mouse splenic lymphocytes or monkey or rabbit PBMC were added at 2 × 105 cells/well along with T1B or T1BT* peptide (0.1 mL/well of 5 μg/mL). Plates were incubated overnight at 37 °C and washed, and species-specific biotinylated anti-cytokine antibody was added (100 μL/well at 2 μg/mL in PBS + 1% BSA). Plates were incubated for one hour at room temperature, washed three times, and 100 μL/well of streptavidin-alkaline phosphatase (R&D Systems, Minneapolis, MN, USA) was added. Plates were incubated for one hour, then washed three times before the addition of 100 μL/well of BCIP/NBT substrate (R&D Systems). Plates were incubated for 15 min in the dark to allow the formation of spots, and the reaction was stopped by rinsing the wells with water. Plates were air-dried overnight, and the spots were counted on an AID Viruspot Reader (AID, Strassberg, Germany).
3
ELISpot Assay for Measuring Cytokine Secretion
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ELISpot plates (Millipore, Watford, UK) were coated overnight with IL-2 capture antibody (R&D Systems, Abingdon, UK), washed, and incubated overnight in blocking buffer (1% BSA in PBS). Cell density was adjusted to 4-6 x 10 6 PBMC/mL, and 100 μL of cells incubated with full length rHuPH20, A33 (positive control), or buffer for 8 days. ELISpot plates were washed, incubated with biotinylated anti-mouse detection antibody (R&D Systems, Abingdon, UK) for 1.5 h at 37°C, followed by incubation with streptavidin-AP (R&D Systems, Abingdon, UK) for 30 min at room temperature. Plates were then incubated with BCIP/NBT substrate (R&D Systems, Abingdon, UK) for 30 min at room temperature. The wells were washed with distilled H 2 0, dried, and scanned on an Immunoscan ® Analyser (Cellular Technology Limited, Cleveland, OH, USA) and spots per well were determined using Immunoscan ® software, Version 5.
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