Human MCAF ELISpot Kit (CAT#: ITS-0322-P167)

96 wells x 6

1.ELISpot Plates.
2.Positive Control.
3.20 X Wash Buffer Concentrated.
4.Concentrated Human MCAF Detection Antibody.
5.Detection Antibody Diluent.
6.Concentrated Streptavidin - AP.
7.Streptavidin-AP Diluent.
8.Substrate Solution.

1.Pipettes with disposable tips, bottles, test tubes and racks, graduated cylinders, absorbent paper, and squirt bottle.
2.37°C CO2 incubator.
3.Deionized or distilled water.
4.Dissection microscope or ELISpot reader.

100 µL

1h

Pre-coated

1.Wash 1 time with Cell Culture Media Fill each well completely with sterile Cell Culture Media. Don't discard until cells are ready to be plated.
2.Prepare Positive Control.
3.Add 2 wells positive control, 2 wells negative control (unstimulated cells), 2 wells background control (sterile cell culture media) and MCAF secreting cells with appropriate concentration to each plate, 100 µL/well. Incubate at 37°C CO2 incubator for 4-48 hours.
4.Prepare 1x Wash Buffer, Human MCAF Detection Antibody solution, and Streptavidin-AP solution.
5.Wash the plate 5 times with 1 x Wash Buffer.
6.Immediately add 100 µL of Human MCAF Detection Antibody to each well of the plate. Cover the plate and incubate 1hour at room temperature (20-25°C).
7.Repeat wash procedure as described in step 5. Wash plate 5 times.
8.Immediately add 100µL of Streptavidin-AP to each well of the plate. Cover the plate and incubate 1hour at room temperature (20-25°C).
9.Repeat wash procedure as described in step 5. Wash plate 5 times.
10.Immediately add 100 µL of Substrate Solution to each well of the plate. Cover the plate and incubate 5-15 minutes at room temperature (20-25°C) in dark.
11.Stop the assay Rinse 5 times with deionized water/distilled water. After final wash, invert plate, and dry by hitting plate onto absorbent paper slightly.
12.Dry plate Wet plates show higher background than completely dry plates. Remove the plastic underdrain from bottom of the plate. Allow the plate dry for 60-90 min at room temperature, or over night at room temperature, or 15-30 min at 37° C in dark. We recommend dry plate over night at room temperature.
13.Quantify spots using a dissection microscope or ELISpot reader.
14.Dried plate can be stored in sealed plastic bag in dark for 6 months.

Ready to use

1.Allow kit reagents and materials to reach room temperature (20-25°C) before use.
2.Do not use kit components beyond their expiration date. Do not substitute reagents from one kit lot to another.
3.The toxicity of the Substrate Solution is not currently known, wear gloves to avoid contact with skin. Follow local, state and federal regulations to dispose of used Substrate Solution.
4.If 20 x Wash Buffer Concentrated is stored at lower temperature (2-8 °C), crystals may form which must be dissolved by warming prior to use.
5.When samples are added to the wells, don't let the pipette tips contact the membrane.

1.Don't let the plate dry during the assay.
2.In order to avoid edge effect don't stack plates during cell incubation.
3.Avoid move the plate during cells incubation period.
4.Don't dry the plate at a temperature higher than 37°C.
5.Spots can't be counted accurately until PVDF membranes were completely dry.

Stored at 2-8 °C.

For Research Grade Use Only.

MCP1; Monocyte chemotactic and activating factor; MCAF; Monocyte chemotactic protein 1; Monocyte secretory protein JE; HSMCR30; Small-inducible cytokine A2; HC11; SMC-CF; GDCF-2; SCYA2; C-C motif chemokine 2; Monocyte chemoattractant protein 1; MCP-1

MCP-1

This gene is one of several cytokine genes clustered on the q-arm of chromosome 17. Chemokines are a superfamily of secreted proteins involved in immunoregulatory and inflammatory processes. The superfamily is divided into four subfamilies based on the arrangement of N-terminal cysteine residues of the mature peptide. This chemokine is a member of the CC subfamily which is characterized by two adjacent cysteine residues. This cytokine displays chemotactic activity for monocytes and basophils but not for neutrophils or eosinophils. It has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, like psoriasis, rheumatoid arthritis and atherosclerosis. It binds to chemokine receptors CCR2 and CCR4.

6347

P13500

Among its related pathways are A-beta Uptake and Degradation and AGE-RAGE signaling pathway in diabetic complications.

1.ELISpot plate coated with human MCAF Capture Antibody.
2.Add stimulated cells or cells and stimulant to the wells and incubate at 37°C in CO2 incubator for a specified period. Secreted human MCAF binds to antibody coated ELISpot plate.
3.Cells and unbounded proteins are washed away.
4.Biotinylated detection antibody is added and binds to the secreted human MCAF.
5.Unbounded proteins are washed away.
6.Streptavidin-AP or Streptavidin-HRP is added and binds to the biotinylated detection antibody.
7.Unbounded proteins are washed away.
8.Substrate Solution is added. A colored precipitate forms and appears as spots at the sites of human MCAF secreting location. Each individual spot representing an individual human MCAF secreting cell.