Ninety-six well plates were incubated overnight at 4°C with SARS-CoV-2 S-2P protein 2 µg /mL (GenScript). On the next day, plates were washed 3 times with PBS and blocked for 2 hours at 37°C with RPMI complete medium (RPMI + 10% fetal calf serum + 1% penicillin/streptomycin + 1% HEPES + 1% L-Glutamine). Freshly isolated splenocytes or bone marrow cells were washed 3 times then resuspended in RPMI complete medium, added to each plate, and serially diluted 2-fold down the plate. After overnight incubation at 37°C, plates were washed extensively with PBS + 0.05% Tween-20 and antibody-secreting cells (ASCs) were detected with biotinylated anti-mouse IgG (1:10,000, Southern Biotech; 1030-08), followed by streptavidin-alkaline phosphatase (1:500, Southern Biotech; 7105-04), and developed with nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (Thermo Scientific). Plates were imaged (Cellular Technologies) and spots were manually counted to determine the number of ASCs.
Nitroblue tetrazolium 5 bromo 4 chloro 3 indolylphosphate
Manufactured by Thermo Fisher Scientific
Nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate is a chromogenic substrate used for the detection and localization of phosphatase enzyme activity in biological samples. It is commonly used in various immunohistochemical and histochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using nitroblue tetrazolium 5 bromo 4 chloro 3 indolylphosphate
1
SARS-CoV-2 Antibody-Secreting Cell Assay
2
SDS-PAGE and Western Blot Analysis of rCTS1
Check if the same lab product or an alternative is used in the 5 most similar protocols
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rCTS1 was either treated with PNGaseF or untreated and subjected to SDS-PAGE under
reducing conditions using 12% polyacrylamide gels at 140 V for 60 minutes (Bio-Rad)
followed by staining with Coomassie Blue dye. For Western blot analysis, electrophoresed
proteins were transferred to a PVDF membrane using a western blot apparatus (Bio-Rad).
After transfer, membranes were blocked in 1% BSA for at least one hour followed by
incubation with anti-CTS1 mouse monoclonal antibody 2F11 at 2 ug/ml in 1% BSA in PBS for 1
hour. Membranes were then washed three times with tris-buffered saline, 0.1% Tween-20
(TBST) followed by addition of peroxidase-conjugated goat anti-mouse IgG antibody (Jackson
ImmunoResearch) at a dilution of 1:5000 for 45 minutes. Membranes were subsequently washed
four times with TBST followed by addition of nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Thermo Scientific) substrate for
development.
reducing conditions using 12% polyacrylamide gels at 140 V for 60 minutes (Bio-Rad)
followed by staining with Coomassie Blue dye. For Western blot analysis, electrophoresed
proteins were transferred to a PVDF membrane using a western blot apparatus (Bio-Rad).
After transfer, membranes were blocked in 1% BSA for at least one hour followed by
incubation with anti-CTS1 mouse monoclonal antibody 2F11 at 2 ug/ml in 1% BSA in PBS for 1
hour. Membranes were then washed three times with tris-buffered saline, 0.1% Tween-20
(TBST) followed by addition of peroxidase-conjugated goat anti-mouse IgG antibody (Jackson
ImmunoResearch) at a dilution of 1:5000 for 45 minutes. Membranes were subsequently washed
four times with TBST followed by addition of nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Thermo Scientific) substrate for
development.
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