Human IFN-gamma/IL-10/Granzyme B FluoroSpotPLUS kit (CAT#: ITS-0322-P134)

10 plates

1.Detection antibodies:anti-IFN-y mAb 7-B6-1-BAM;anti-IL-10 mAb 12G8, biotinylated;anti-Granzyme B mAb MT28-WASP.
2.Fluorophore conjugates: Anti-BAM-490;SA-550;anti-WASP-640.
3.Co-stimulator:anti-CD28 mAb CD28A.
4.Positive control:anti-CD3 mAb CD3-2
5.Fluorescence enhancer 2:Ready-to-use Fluorescence enhancer-II.
6.Pre-coated plates:Plates pre-coated with:mAbs 1-D1K,9D7 and MT34B6.

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 5-15minutes 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 (7-B6-1),Biotinylated detection mAb (12G8),WASP-conjugated detection mAb (GB11),Anti-BAM-490,SA-550,Anti-WASP-640,anti-CD3 mAb,Anti-CD28 mAb (CD28-A),Fluorescence enhancer,Pre-coated FluoroSpot plates (mAbs 1-D1K, 9D7 and GB10)

Anti-CD28 mAb provides a co-stimulatory signal to antigen-specific responses by binding to CD28 on T cells. Addition of an anti-CD28 mAb together with antigen(step B1) can be usedto enhance antigen-specific responses. However, if the concentration of anti-CD28 mAb is toohigh, non-specific cytokine secretion may be elevated. Anti-CD28 can be used to circumventcapture effects, which can occur when different capture antibodies are coated in the same well,where absorption of one cytokine may negatively affect the secretion of another cytokine.

PBS for washing and dilution should be filtered (0.3 um) for optimal results.Although possible to use, we donot 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.

3002/ 3458/ 3586

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.