Human IL-1beta/IL-6/TNF-alpha FluoroSpotPLUS kit (CAT#: ITS-0322-P135)

10 plates

1.Detection antibodies:anti-IL-1β mAb 7P10-BAM;anti-IL-6 mAb 39C3, biotinylated;anti-TNF-a mAb MT20D9-WASP.
2.Fluorophore conjugates: Anti-BAM-490;SA-550;anti-WASP-640.
3.Stimulus:LPS.
4.cell incubation time:overnight.

200 µL

2h

Pre-coated

1.Remove the plate from the sealed package and wash the plate three times with sterile PBS (200 μl/well).
2.Block/ condition the plate by adding cell incubation medium containing 10% fetal calf serum (200 ul/well). Incubate for at least 30 minutes at room temperature.
3.Remove the medium and add the stimuli followed by the cell suspension. Cells and stimuli can also be mixed before addition to the plate.
4.Incubate the cells in the plate at 37°C in a humidified incubator with 5% CO2.
5.Remove the cells by emptying the plate and then wash the plate five times with PBS (200ul/well).
6.Dilute the detection antibodies in PBS containing 0.1 % BSA (PBS-0.1% BSA) according to the table above. If more than one analyte is analyzed, dilute the detection antibodies in the same tube. Add 100 μl/well and incubate for 2 hours at room temperature.
7.Wash the plate five times with PBS (200 ul/well).
8.Dilute the fluorophore-conjugates in PBS-0.1 % BSA to the concentrations as shown in the table above. If more than one analyte is analyzed, dilute the conjugates in the same tube. Add 100 μl/well and incubate for 1 hour at room temperature. Protect the plate from light throughout the assay.
9.Wash the plate five times with PBS (200 ul/well).
10.Empty the plate, add Fluorescence enhancer (50 ul/well), and leave for 5-15 minutes at room temperature.
11.Empty by flicking the plate to remove the Fluorescence enhancer.
12.Remove the underdrain (the soft plastic drain under the plate). Dry the plate protected from light. The plate should be completely dry before analysis. Store plate in the dark at room temperature.
13.Spot analysis is performed with an automated FluoroSpot reader equipped with filters for the fluorophores used. Filters should have high specificity to avoid bleed-through artifacts.

human

BAM-conjugated detection mAb (7P10), Biotinylated detection mAb (39C3), WASP-conjugated detection mAb (MT20D9), Anti-BAM-490, SA-550, Anti-WASP-640, Fluorescence enhancer, Pre-coated FluoroSpot plates (mAbs 13A5, MT175 and MT25C5), Positive control (LPS)

An automated FluoroSpot reader with separate filters is required for analysis. The reader should be equipped with filters for the fluorophores used. Filters should be selective for the specific wavelengths to avoid bleed-through artifacts. In order to obtain accurate measurements of cells secreting multiple analytes, the automated reader must be able to create an overlay analysis from the filters. To avoid fading, the plates should be kept dark and should preferablybe analyzed within 1 week of development.

PBS for washing and dilution should be filtered (0.3 um) for optimal results. Although possible to use, we do not recommend the inclusion of Tween or other detergents in the washing and incubation buffers.

4°C-8°C

Plates should be kept at room temperature.

The expiry date indicates how long unopened products, stored according to instructions, are recommended for use.

The serum should be selected to support cell culture and give low background staining. We recom-mend the use of fetal calf serum. Alternatively serum-free medium evaluated for cell culture can beused.

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

3553/ 3569/ 7124

Our FluoroSpotPLUS kits include pre-coated plates ready to use. Monoclonal antibodies (mAbs) are coated onto a PVDF membrane in a 96-well plate. If several analytes are analyzed, mAbs with different specificities are coated. Detection of cells secreting single or multiple analytes is made possible using biotinylated and/or tag-labeled detection mAbs. The detection step is visualized and amplified by specific fluorophore-conjugated reagents and the resulting spots are analyzed in an automated reader.