| Catalog No. | Size | Price (USD) |
| EK50349A-1 | 1 Kit (96 Tests) | $370.28 |
| EK50349A-10 | 10 Kit (96 Tests) | $2,666.00 |
General Information
| Intended Use | The kit applies for the detection of mouse TNF-α at the cellular level by counting TNF-α-secreting cells. |
| PRINCIPLE OF THE ASSAY | The enzyme-linked immunospot (ELISPOT) assay employs a sandwich ELISA-based methodology. A 96-well polyvinylidene difluoride (PVDF) membrane plate, pre-coated with mouse TNF-α-specific monoclonal antibodies, captures mouse TNF-α secreted by activated cells. Following cell removal and washing, biotin-conjugated secondary antibodies targeting TNF-α are added to form antibody-antigen-antibody "sandwich" complexes. Subsequent incubation with streptavidin-alkaline phosphatase (ALP) conjugates enables biotin-mediated binding. Addition of chromogenic substrate culminates in the formation of insoluble spots at the membrane interface, with each spot representing an individual TNF-α-secreting cell. Spot quantification is achieved by automated ELISPOT analysis or manual counting using a microscope. |
| KIT CONTENTS AND STORAGE | Please use the kit before the expiration date. Components Amount Preparation instructions storage Pre-coated microplate 1plate (96 tests) Once removed from vacuum bag, the plate should be used as soon as possible. Vacuum storage can store at 2-8℃until expiration date. Detection Antibody 1 vial Dilute the detection antibody 1:370 in PBS containing 0.5% fetal calf serum (PBS-0.5% FBS) before use. Dilute fresh as needed. Primary liquid are stable at 2 - 8℃ until expiration date. To be reconstituted, the working fluid is used within the working day and discard. So dilute fresh as needed. Streptavidin-ALP 1 vial Dilute the Streptavidin-ALP 1:170 in PBS-0.5% FBS. BCIP/NBT-plus Dilution 1 bottle BCIP/NBT-plus Solution A and the Dilution should be mixed together in a 1:250 volume, followed by the addition of Solution B in the same volume as Solution A and mixed gently. The volume ratio of Solution A:Dilution:Solution B is 1:250:1. For example: pipette 40 μL of Solution A into 10 mL of the Dilution buffer, mix thoroughly, then add 40 μL of Solution B and mix gently. Dilute fresh as needed. BCIP/NBT-plus Solution A 1 bottle BCIP/NBT-plus Solution B 1 bottle |
| ASSAY PROCEDURE | 1. Plate Set-up (sterile conditions) Remove the plate from the sealed package and wash 4 times with sterile PBS (200 µl/well). Condition the plate with sterile RPMI-1640 culture media (200 µl/well) containing 10% fetal calf serum (FBS) as used for the cell suspensions, Incubate for at least 30 minutes at room temperature. 2. Incubation of cells in plate (sterile conditions) Empty the plate, then add stimulus followed by the 100 µl cell suspensions to each well. Theconcentration of cell and stimulus should be adjusted according to the actual situation.The recommended group settings are as follows. a. Positive control: Fewer cells such as 1×10 5 cells/wellare needed for polyclonal activators, e.g., LPS (0.1-0.5 µg/ml). b. Negative control: The cell concentration is kept consistent with the experimental wells in the absence of stimulus. c. Background control: No cells, no positive stimulus, add to the medium. d. Experimental group: The recommended cell number per well is 1.5–2.5 × 10 5 , and add your own stimulus as required by the experiment (prepare the stimulus using culture medium). Note: It is recommended to set duplicate wells for samples to be tested. Put the plate in a 37℃ and 5% CO 2 incubator for 18-48 hours.Do not move the plate during this period and it is recommended to wrap the plate in aluminum foil to avoid evaporation. 3. Detection of spots Note: Use PBS containing 0.5% FBS (PBS-0.5% FBS) for dilution ofthe Biotin-conjugated antibody antibody andStreptavidin-ALP. The PBS should be filtered (0.2 um) for optimal results.Avoid the inclusion of Tween or other detergents in the washing and incubation buffers. Remove the cells by emptying the plate and wash 5 times with PBS, 200 µl/well. Note: Soak in the PBS for 1 minute to ensure thorough cleaning. The same requirement applies to all subsequent washing steps. Dilute the detection antibody 1:370 in PBS-0.5% FBS. Add 100 µl/well and incubate for 2 hours at room temperature. Note: Diluted detection antibody can be filtered (0.2 µm) to reduce the risk of unspecific background. Empty the plate and wash 5 times with PBS, 200 µl/well. Dilute the Streptavidin-ALP 1:170 in PBS-0.5% FBS and add 100 µl/well. Incubate for 1hour at room temperature. Empty the plate and wash 5 times with PBS, 200 µl/well. Filter the ready-to-use substrate solution (BCIP/NBT-plus) through a 0.45 µm filter and add 100 µl/well. Develop until distinct spots emerge in positive wells (usually 5-30 minutes). Note: Too long the color development time will cause the background color in the experimental well to become darker. Stop color development by washing extensively in tap water. If desirable, remove the underdrain (the soft plastic under the plate) and rinse the underside of the membrane. Leave the plate to dry. Plates should be completely dry before analysis. Inspect and count spots in an ELISpot reader or in a microscope. Note：Do not dry the microplate at atemperature higher than 37°C; this may cause cracking of the membrane filters. For the option to re-analyze later, store plate in the dark at room temperature. |
| TROUBLESHOOTING GUIDE | Problem Possible reason Solution Dark background of the PVDF membrane *Plate is insufficiently washed * The membrane is wet *Increase the washing times to ensure soaking time *Microplates should be analyzeduntil the PVDF membranes are completely dry Spots appear in the negative control wells *Endotoxins or other contaminants in the medium, serum, or DMSO may activate immune cells *The cells are contaminated *Cell viability is low *Use endotoxin-free reagents *Pay attention to aseptic procedures in the step of Plate Set-up and Incubation of cells in plate *Ensure high viability (recommended viability > 90%) as well as functionality of the cell sample Spots are irregular in shape and severely clumped *Cell disruption *Cell aggregated *Optimize the cell separation process to ensure cell activity *The cell suspension is thoroughly mixed toensure a unicellular state Positive Control spots were low and fuzzy *Enzyme activity is decreased *The temperature of the Substrate reagent is low and oxidized *The titer of the stimulus is low *Increase enzyme concentration *Balance the color reagent to room temperature before use, do not use reagents that have oxidized precipitation *Increase the concentration of stimulus The spots are unevenly positioned and have an edge effect *Shake the cell plate after plating, causing cell displacement *Add the stimulus too vigorously to flush the cell suspension *Stack the plates in the incubator *After plating, try not to shake the plate, and gently pan it into the incubator *Use the method of adding the stimulus first and then the cells *Place each plate individually on the shelf to allow an even distribution of heat to each microwell Not all spots are counted in the crowded well despite algorithm and parameter adjustments *Spots are confluent. Single spots cannot be discriminated *Decrease the number of cells added per well |
| Performance | Sino Competitor M The statistical histogram and representative plate images of mTNF-α ELISpot assays. Splenocytes isolated from BALB/c mice were plated in graded densities (5×10 4 –2.5×10 5 cells/well) and stimulated with Lipopolysaccharide (LPS) for 20 h. Wells containing cells without stimulation were designated as the negative control, while those containing all reagents in the absence of cells served as the background control. |
Synonyms: DIF; Tnf; TNF-a; TNF-alpha; Tnfa; TNFalpha; Tnfsf1a; TNFSF2; Tnlg1f
Image / Experimental Information
| Image 1 Alt Text | EK50349A-mTNF-Sino-Mabtech |
| Image 1 URL | Source image |
Background Information
| Full Name | tumor necrosis factor |
| Description | Tumor necrosis factor alpha (TNF-alpha), also known as TNF, TNFA or TNFSF2, is the prototypic cytokine of the TNF superfamily, and is a multifunctional molecule involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. Two receptors, TNF-R1 (TNF receptor type 1; CD120a; p55/60) and TNF-R2 (TNF receptor type 2; CD120b; p75/80), bind to TNF-alpha. TNF-alpha protein is produced mainly by macrophages, and large amounts of this cytokine are released in response to lipopolysaccharide, other bacterial products, and Interleukin-1 (IL-1). TNF-alpha is involved in fighting against the tumorigenesis, thus, is regarded as a molecular insight in cancer treatment. |
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title: Neuroblast Differentiation-Associated Protein Derived Polypeptides: AHNAK(5758-5775) Induces Inflammation by Activating Mast Cells via ST2.
authors: Xiangjin Song; Lei Zhang; ...; Songmei Geng
journal: Immunological investigations
date: 2023-02-02
pmid: 36511894
doi: 10.1080/08820139.2022.2151368
cited_by_count: 4
techniques: Enzyme-linked Immunosorbent Assay
bioz_score: 91
