Human GM-CSF ELISpot Kit (CAT#: ITS-0322-P25)

1x96 Wells

1.ELISpot Plates
2.Positive Control
3.20 X Wash Buffer Concentrated
4.Human GM-CSF Detection Antibody
5.Concentrated Streptavidin - AP
6.Streptavidin - AP Diluent
7.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.
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 GM-CSF secreting cells with appropriate concentration to each plate, 100 µL/well. Incubate at 37°C CO2 incubator for 4-48 hours. Each researcher should determine the optimal incubation time based on the characteristics of the cell.
4.Prepare 1x Wash Buffer and Streptavidin - AP solution.
5.Wash the plate 5 times with 1 x Wash Buffer.
6.Immediately add 100 µL of Human GM-CSF 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 100 µL 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.

Human

NP_000749.2

Liquid

1.For research use only not to be used as a diagnostic test.
2.Do not eat, drink, smoke or apply cosmetics where kit reagents are used.
3.Cover or cap all reagents when not in use.
4.Do not mix or interchange reagents between different lots.
5.Do not use reagents beyond the expiration date of the kit.
6.When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.
7.BCIP/NBT buffer is potentially carcinogenic and should be disposed of appropriately, caution should be taken when handling this reagent, always wear gloves.

1.Handling of reagents, serum or plasma specimens should be in accordance with local safety procedures.
2.When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles labels.
3.All reagents should be warmed to room temperature before use.
4.Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross contamination.
5.Use a clean plastic container to prepare the washing solution.
6.Thoroughly mix the reagents and samples before use by agitation or swirling.

All reagents provided are stored at 4°C.

Refer to the expiration date on the label.

Select online data sheet information is drawn from bioinformatics databases, occasionally resulting in ambiguous or non-relevant product information. It is the responsibility of the customer to review, verify, and evaluate the information to make sure it matches their requirements before purchasing the kit. Our ELISA Kit assays are dynamic research tools and sometimes they may be updated and improved.

For Research Use Only. Not for use in diagnostic procedures.

GM-CSF; Granulocyte-macrophage colony-stimulating factor; CSF; GMCSF; Molgramostin; Colony-stimulating factor; Sargramostim

GMCSF; MGC131935; MGC138897

The protein encoded by this gene is a cytokine that controls the production, differentiation, and function of granulocytes and macrophages. The active form of the protein is found extracellularly as a homodimer. This gene has been localized to a cluster of related genes at chromosome region 5q31, which is known to be associated with interstitial deletions in the 5q- syndrome and acute myelogenous leukemia. Other genes in the cluster include those encoding interleukins 4, 5, and 13.

1437

P04141

Among its related pathways are PEDF Induced Signaling and Hematopoietic Stem Cell Differentiation.

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