Mouse IgM ELISpotBASIC kit (ALP) (CAT#: ITS-0322-P111)

4 plates

1.Vial 1 (green top):Monoclonal antibody MT6A3 (1200 μl);Concentration: 0.5 mg/ml.
2.Vial 2 (yellow top):Biotinylated monoclonal antibody MT9A2 (100 μl);Concentration: 0.5 mg/ ml.
3.Vial 3 (white top):Streptavidin Alkaline Phosphatase (50 ul).
4.Vial 4 (black top):Polyclonal activator: R848 (100 ul);Concentration:1 mg/ ml.
5.Lyophilized recombinant mouse IL-2(0.5 ug).

200 µL

2h

Pre-coated

1.Dilute the coating antibodies (MT6A3) to 15 ug/ml in sterile PBS, pH 7.4.
2.Remove the ELISpot plate from the package and if using a PVDF plate, pre-wet the membrane by adding ethanol.
3.Wash plate 5 times with sterile water, 200 ul/well.
4.Add 100 ul/well of the antibody solution and incubate overnight at 48°C.
5.Remove excess antibody and wash plate 5 times with sterile PBS, 200 ul/well.
6.Add 200 ul/well of medium containing 10% of the same serum as used for the cell suspensions Incubate for at least 30 minutes at room temperature.
7.Remove the medium and add the stimuli followed by the cell suspension. Alternatively cells and stimuli can be mixed before addition to the plate.
8.Put the plate in a 37°C humidified incubator with 5% CO, and incubate for 18-48 hours.
9.Remove the cells by emptying the plate and wash 5 times with PBS, 200 ul/well.
10.Dilute the detection antibody(MT9A2-biotin) to 0.05 ug/ml in PBS containing 0.5% fetal calfserum(PBS-O.5%FCS).Add 100 ul/well and incubate for 2 hours at room temperature.
11.Wash plate as above (step C1).
12.Dilute the Streptavidin-ALP(1:1000) in PBS-0.5% FCS and add 100 ul/well. Incubate for 1 hour at room temperature.
13.Wash plate as above (step C1).
14.Filter the ready-to-use substrate solution(BCIP/NBT-plus) through a 0.45 um filter and add100 ul/well. Develop until distinct spots emerge.
15.Stop color development by washing extensively in tap water. If desirable, remove the underd-rain (the soft plastic under the plate) and rinse the underside of the membrane.
16.Leave the plate to dry.Inspect and count spots in an ELISpot reader or in a dissection micro-scope.
17. Store plate in the dark at room temperature.

mouse

Capture mAb (MT6A3),Biotinylated detection mAb (MT9A2),Streptavidin-ALP,R848,Recombinant mouse IL-2

We recommend the use of PVDF-based membrane plates. Maximal antibody binding capacity of these platesis obtained by a brief treatment with ethanol.

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. R848 and IL-2 shoud be stored frozen at -20°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.

AI326478, Igh-6, Igh-M, Igh6, Igm, muH

16019

Antibodies from all secreting B cells are captured by anti-IgM antibodies coated on the ELISpot plate. Cells producing specific antibody are then visualised by the addition of biotinylated antigen followed by Streptavidin-enzyme and substrate.