膜杰作
Star Staining
组分（Materials Provided）
| ID | Components | Size |
| RSP010-C01 | Pre-coated Anti-IL-4 Antibody Microplate | 1 plate |
| RSP010-C02 | Positive Stimulus | 150 μg |
| RSP010-C03 | Biotin-Anti-IL-4 Antibody | 50 μL |
| RSP010-C04 | Streptavidin-HRP | 50 μL |
| RSP010-C05 | Washing Buffer (10×) | 50 mL |
| RSP010-C06 | Dilution Buffer (1×) | 50 mL |
| RSP010-C07 | AEC Dilution | 25 mL |
| RSP010-C08 | AEC Solution A (20×) | 0.8 mL |
| RSP010-C09 | AEC Solution B (20×) | 0.8 mL |
| RSP010-C10 | AEC Solution C (20×) | 0.8 mL |
背景（Background）
IL-4 is known to be the quintessential regulatory cytokine, playing a role in a vast number of immune and non-immune functions. This cytokine is commonly secreted by type 2 helper T (TH2) cells and follicular helper T (TFH) cells after antigenic sensitization. The Mouse IL-4 ELISpot assay is designed for the detection of mouse IL-4 secreting cells at the single cell level and can be used to quantitate the frequency of mouse IL-4 secreting cells. ELISpot assays are highly reproducible and sensitive, and can be used to measure responses with frequencies well below 1 in 100,000. ELISpot assays do not require prior in vitro expansion of T cells and they are suitable for high-throughput analysis using only small volumes of primary cells. As such, ELISpot assays are useful tools for research in vaccine development, cytokine secretion and the monitoring of various clinical trials.
应用说明（Application）
This kit is used to detect mouse IL-4 at the cellular level.
It is for research use only.
重构方法（Reconstitution）
Please see Certificate of Analysis for details of reconstitution instruction and specific concentration.
存储（Storage）
1. Unopened kit should be stored at 2℃-8℃ upon receiving.
2. Find the expiration date on the outside packaging and do not use reagents past their expiration date.
3. Please use it up once you opened it.
原理（Assay Principles）
ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. The Kit consists of Pre-coated Anti-IL-4 Antibody Microplate, Positive Stimulus, Biotin-Anti-IL-4 Antibody, Streptavidin-HRP, AEC and buffers.
Your experiment will include 6 simple steps:
a) A monoclonal antibody specific for mouse IL-4 has been pre-coated onto a polyvinylidene difluoride (PVDF)-backed microplate.
b) Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37 °C CO2 incubator for a specified period of time.During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted IL-4.
c) After washing away any cells and unbound substances, a biotinylated antibody specific for mouse IL-4 is added to the wells. Following a wash to remove any unbound biotinylated antibody, HRP conjugated to streptavidin is added.
d) Wash the plate and add the Streptavidin-HRP diluted by Dilution Buffer to the plate.
e) Wash the plate and add AEC. A reddish-brown colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual IL-4 secreting cell.
f) The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.
典型数据-Typical Data Please refer to DS document for the assay protocol.
2.5×10⁵ cells/well Mouse Spleen Cells were stimulated with 18 μg/mL Positive Stimulus and cultured at 37℃ and 5%CO2 for 20h, 228 spots were produced. The following example data is for reference only.
