Mouse IL-10 ELISA Kit (MOFI00054)- High Sensitivity

Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Mouse IL-10 ELISA Kit
Product Code:	MOFI00054
Size:	96 Assays
Alias:	IL-10, Interleukin 10, IL10, IL10A, CSIF, TGIF
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse IL-10 concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Mouse Il-10 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse IL-10 in samples.


Matrix	Recovery Range (%)	Average (%)
serum (n=5)	85-101	94
EDTA plasma (n=5)	89-105	97
UFH plasma (n=5)	88-100	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse IL-10 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.


Sample	1:2	1:4	1:8
Serum (n=5)	89-100%	86-104%	87-105%
EDTA plasma (n=5)	84-101%	84-93%	88-99%
UFH Plasma (n=5)	83-98%	80-94%	85-96%

Intra Assay

CV < 8%

Inter Assay

CV < 10%

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate (SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Protocol

*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

IL-10 Background

Interleukin 10

Interleukin-10 (IL-10) is a vital immunoregulatory cytokine that plays a significant role in the immune system's response to infections and inflammatory processes. It is a part of the larger interleukin family, which consists of various cytokines that are involved in cell signaling and communication during immune responses.

Interleukin 10 Gene

In mice, the gene encoding IL-10 is called Il10. It is located on chromosome 1 and contains 5 exons. In humans, the IL-10 gene is found on chromosome location 1q32.1 and contains 7 exons.

IL 10 Function

The primary function of IL-10 is to modulate the immune response and control inflammation. It exerts both immunosuppressive and anti-inflammatory effects on various immune cells, including T cells, B cells, macrophages, dendritic cells, and natural killer cells. Some key function are :

Immunosuppression: IL-10 inhibits the production of pro-inflammatory cytokines, such as IL-1, IL-6, and TNF-alpha, which are responsible for promoting inflammation. This helps in preventing excessive and harmful inflammatory responses.
Anti-inflammatory: IL-10 suppresses the activity of certain immune cells, particularly Th1-type T cells, which are known to drive inflammatory responses. By downregulating these cells, IL-10 helps to maintain immune homeostasis.
Tissue Repair and Tolerance Induction: IL-10 also plays a role in tissue repair and tolerance induction. It promotes the generation of regulatory T cells (Tregs), which help in dampening immune responses and preventing autoimmune reactions.

IL 10 Signalling

The Interleukin-10 (IL-10) signaling pathway involves the binding of IL-10 to its receptor, forming a heterodimeric complex of IL-10R1 and IL-10R2 subunits. This triggers the activation of Janus kinases (JAKs), particularly JAK1 and Tyk2, which phosphorylate STAT3 proteins. The phosphorylated STAT3 proteins form dimers and translocate to the nucleus, where they bind to STAT response elements (SREs) in the promoter regions of target genes. This leads to the transcriptional regulation of anti-inflammatory molecules, including IL-10 itself. Consequently, the IL-10 signaling pathway suppresses pro-inflammatory cytokines (e.g., IL-1, IL-6, TNF-alpha) and inhibits the activities of immune cells promoting inflammation, such as Th1-type T cells and macrophages. The pathway's immunomodulatory effects help to maintain immune homeostasis and control excessive or harmful inflammation. Dysregulation of the IL-10 pathway has been linked to various immune-related disorders and inflammatory conditions, highlighting its significance as a therapeutic target to modulate inflammation and promote immune balance.

IL-10 Clinical Significance

Clinical Significance: IL-10 has significant clinical significance in various medical conditions:

Inflammatory Disorders: Dysregulation of IL-10 has been implicated in various inflammatory disorders, such as inflammatory bowel disease (IBD), rheumatoid arthritis, and psoriasis. Low levels of IL-10 can lead to uncontrolled inflammation, while high levels may contribute to immunosuppression and increased susceptibility to infections.
Infectious Diseases: IL-10 plays a dual role in infectious diseases. It can be beneficial by controlling excessive inflammation during infections, but on the other hand, some pathogens exploit IL-10 to evade the immune system, leading to persistent infections.
Cancer: In certain cancers, IL-10 may promote tumor growth by suppressing the anti-tumor immune response. However, it can also have anti-tumor effects in some cases by modulating the tumor microenvironment.
Therapeutic Potential: Due to its immunoregulatory properties, IL-10 has been explored as a potential therapeutic target. Researchers have investigated the use of IL-10 in various autoimmune diseases and have also studied IL-10-based therapies for cancer treatment.

Mouse IL-10 ELISA Kit FAQs

Q: What is the purpose of the Mouse IL-10 ELISA kit ?

IL-10 is a crucial immunoregulatory cytokine that plays a central role in immune responses, inflammation, and tolerance induction. By using an IL-10 kit, researchers can accurately determine the concentration of IL-10 in different samples, such as serum, plasma, cell culture supernatants, and tissue homogenates. This information is invaluable in studying the role of IL-10 in various physiological and pathological processes, including infectious diseases, autoimmune conditions, inflammatory disorders, cancer, and other immune-related disorders.

Q: What type of samples can be used with the Mouse IL-10 ELISA kit?

The Mouse IL-10 ELISA kit is designed to work with a variety of biological samples, including but not limited to serum, plasma, cell culture supernatants, and tissue homogenates. Before running the assay, it is essential to follow the kit's specific instructions for sample preparation and dilution to ensure optimal results.

Q: Can the kit be used for diagnostic purposes?

The Mouse IL-10 ELISA kits are primarily intended for research purposes and not for diagnostic or clinical use. These kits are used in laboratories for scientific investigations and experiments to study the levels of specific proteins in various samples.

Q: Can this kit be used for other species or proteins?

ELISA kits are typically designed and optimized for specific target proteins and species. While the Mouse IL-10 ELISA kit is intended for quantifying mouse IL-10, there might be similar ELISA kits available for other species or different proteins. Researchers should use the appropriate kit for their specific target protein and species of interest.

Q: Where can I find additional technical support or assistance with the kit?

For any technical inquiries or assistance regarding the Ukit, you can reach out to our team. They will be available to answer your questions and provide the necessary guidance to ensure a successful experiment.

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Mouse IL-10 ELISA Kit

Description