Mouse Granzyme B ELISpot Development Module, 5 Plate (CAT#: ITS-0322-P163)

1 Kit (for 5 Plates)

1.Mouse Granzyme B Capture Antibody.
2.Mouse Granzyme B Biotinylated-conjugated Detection Antibody.

1.ELISpot Blue Color Module or equivalent.
2.PBS - 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered.
3.Wash Buffer - 0.05% Tween® 20 in PBS.
4.Blocking Buffer - 1% BSA, 5% Sucrose in PBS.
5.Reagent Diluent - 1% BSA in PBS, pH 7.2 - 7.4, 0.2 µm filtered.
6.2 °C - 8 °C refrigerator.
7.37 °C CO2 incubator.
8.Positive Control - Use Recombinant Human IgG B Cell ELISpot Development Modules or cells known to secrete Human IgG B Cell ELISpot Development Modules.
9.96-well plates - Nitrocellulose-bottom plates, PVDF-bottom Immunospot® plates, or flat-bottom polystyrene Immulon® ELISA plates.
10.Multi-channel pipette, squirt bottle, manifold dispenser, or automated microplate washer.
11.Dissection microscope or an automated ELISpot Reader.
12.Deionized H2O.

100 µL

3 hours 35 mins to 4 hours 50 mins**

Pre-coated

1.Capture Antibody Concentrate: Reconstitute with 1 mL of PBS. After reconstitution, store at 2-8 °C for up to 60 days or aliquot and store at -20 °C to -70 °C in a manual defrost freezer for up to 6 months.
2.Detection Antibody Concentrate: Reconstitute with 1 mL of Reagent Diluent. After reconstitution, store at 2-8 °C for up to 60 days or aliquot and store at -20 °C to -70 °C in a manual defrost freezer for up to 6 months.

96-well microplate, sold separately

Avoid inhalation, contact with eyes, skin and clothing. Avoid the formation of dust and aerosols. Use in a well-ventilated area. Keep away from sources of ignition. Avoid prolonged or repeated exposure.

Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Store the unopened product at -20 to -70 °C.

Store in cool, well-ventilated area. Keep away from direct sunlight. Keep container tightly sealed until ready for use.

For Research Grade Use Only.

C11; CCPI; CGL1; CGL-1; CSPB; CSP-B; CTLA1; CTLA-1; CTSGL1; Fragmentin-2; Granzyme B; Granzyme-2; GranzymeB; GRB; GrzB; GZMB; HLP; SECT

AI553453, CCP-1/C11, CCP1, Ctla-1, Ctla1, GZB

Granzyme family serine proteases are stored in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes contain one S1 protease domain and are synthesized as preproproteins. Granzymes are released toward pathogen infected or transformed target cells during cellular immune reactions. Target cell entry by granzymes is through perforin channels. Granzymes trigger apoptosis by caspase-dependent and -independent mechanisms. There are five granzymes (A, B, G, H and K) in human and many more in mouse and rat.

14939

P04187

Among its related pathways are Activation of BH3-only proteins and Allograft rejection.

1.Calculate the total volume of Capture Antibody needed and dilute to the working concentration in PBS.
2.Immediately add 100 µL of the diluted Capture Antibody or antigen per well. Cover the microplate with the lid and incubate overnight at 2-8 °C.
3.Aspirate each well and wash 3 times with Wash Buffer or PBS (350 µL/well) using a squirt bottle, manifold dispenser, or autowasher. After the final wash, remove any remaining liquid by inverting the microplate and blotting it against a clean paper towel.
4.Block membranes by adding 200 µL of Blocking Buffer to each well. Incubate for 2 hours at room temperature.
5.Aspirate or decant Blocking Buffer. Rinse with the same media in which the cells will be cultured.
6.Aspirate culture media from the microplate, and immediately fill the appropriate wells with 100 µL of culture media containing mouse Granzyme B secreting cells. Incubate at 37°C in a 5% CO2 incubator.
7.Wash the microplate 4 times with Wash Buffer. Remove any remaining Wash Buffer by inverting the microplate and blotting it against a clean paper towel.
8.Calculate the total volume of Detection Antibody needed and dilute to the working concentration in Reagent Diluent.
9.Add 100 µL of the diluted Detection Antibody or biotinylated antigen per well. Cover the microplate with the lid and incubate overnight at 2-8 °C.
10.Aspirate Detection Antibody or biotinylated antigen and wash as described in step 3. Microplates are now ready for color development.