H-2Dd antibody

Catalog No. ABIN114289

Product Details anti-H-2Dd Antibody

Target: H-2Dd (MHC Class I H-2Dd)

Reactivity: Mouse

Host: Mouse

Clonality: Monoclonal

Conjugate: This H-2Dd antibody is un-conjugated

Application: Cytotoxicity Test (CyTox)

Specificity: This mAb is specific for cells expressing the H-2D antigen coded for by the d haplotype. The reaction pattern of this antibody with a panel of inbred and recombinant haplotypes demonstrates that the antibody detects a private determinant (H-2.4) of the H-2Dd antigen. This antibody can be used to quantitate or eliminate cells bearing H-2Dd (H-2.4) antigen from the appropriate strains of mice.

Purification: Protein G Chromatography

Immunogen: Recipient: C3H/HeJ Donor: B6XDBA/2 spleen cells Fusion Partner: SP2/0.Ag14

Clone: 34-5-8S

Isotype: IgG2a

Application Details

Application Notes: Cytotoxicity assay. Functional testing.
Other applications not tested.
Optimal dilutions are dependent on conditions and should be determined by the user.

Protocol: RECOMMENDED METHOD FOR DEPLETING A CELL POPULATIONOF H-2Dd BEARING LYMPHOCYTES: 1. Prepare a cell suspension from the appropriate tissue in Cytotoxicity Medium1 orequivalent. Remove red cells and dead cells (where necessary) by purification of viablelymphocytes on Lympholyte®-M density cell separation medium. After washing, adjust thecell concentration to 1x10e7 cells per ml in Cytotoxicity Medium. 2 Add the antibody to afinal concentration of 1: 80 and mix. Alternatively, pellet the cells and resuspend inantibody diluted 1: 80 in Cytotoxicity Medium. 3. Incubate for 60 minutes at 4°C. 4. Centrifuge to pellet the cells and discard the supernatant. 5. Resuspend to the original volume in Low-Tox®-M Rabbit Complement, diluted to theappropriate concentration in Cytotoxicity Medium. (Recommended concentration includedwith each batch of Low-Tox®-M Rabbit Complement. )6. Incubate for 60 minutes at 37°C. 7. Monitor for percent cytotoxicity at this stage, before further processing. For this purposeremove a small sample from each tube, dilute 1: 10 with medium, and add 1/10 volume of1% Trypan Blue. After 3-5 minutes, score live versus dead cells in a haemacytometer. 8. For functional studies, remove the dead cells from the treated groups before furtherprocessing, particularly if the treated cells are to be cultured. This can be done by layeringthe cell suspension separation medium and centrifuging at room temperature as per theinstructions provided. Live cells will form a layer at the interface, while the dead cellspellet. The interface can then be collected and washed in Cytotoxicity Medium before beingresuspended in the appropriate medium for further processing. Alternately, the cells canbe washed and resuspended in the appropriate medium for further processingimmediately after Step 6. , provided that the dead cells will not interfere with subsequentassays. RECOMMENDED METHOD FOR DETERMINING PERCENT OF H-2Dd BEARING CELLS IN A POPULATION: 1. Prepare a cell suspension from the appropriate tissue in Cytotoxicity Medium1 orequivalent. Remove red cells and dead cells (where necessary) by purification of viablelymphocytes on Lympholyte®-M density cell separation medium. After washing, adjust thecell concentration to 1x10e6 cells per ml in Cytotoxicity Medium. 2. Add the antibody to a final concentration of 1: 80 and mix. 3. Incubate for 60 minutes at 4°C. 4. Centrifuge to pellet the cells and discard the supernatant. 5. Resuspend to the original volume in Low-Tox®-M Rabbit Complement3 diluted to theappropriate concentration in Cytotoxicity Medium. (Recommended concentration includedwith each batch of Low-Tox®-M Rabbit Complement. 6. Incubate for 60 minutes at 37°C. 7. Place on ice. 8. Add Trypan Blue, 10% by volume of 1% Trypan Blue (w/v) added 3-5 minutes beforescoring works well. Score live versus dead cells in a haemacytometer. Cytotoxic Index (C. I. )can be calculated as shown in figure 1. NOTES: 1. Cytotoxicity Medium is RPMI-1640 with 25mM Hepes buffer and 0. 3% bovine serumalbumin (BSA). BSA is substituted for the conventionally used fetal calf serum (FCS)because we have found that many batches of FCS contain complement dependentcytotoxins to mouse lymphocytes, thus increasing the background killing in the presenceof complement. We recommend that cells not be exposed to FCS prior to or duringexposure to antibody and complement. Some batches of BSA also contain complement

Restrictions: For Research Use only

Handling

Concentration: 1.0 mg/mL

Buffer: PBS and 0.02 % NaN3

Preservative: Sodium azide

Precaution of Use: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: Avoid repeated freezing and thawing.

Storage: 4 °C/-20 °C

Storage Comment: Store the antibody undiluted at 2-8 °C for one month or (in aliquots) at -20 °C for longer.

Target Details for H-2Dd

Target: H-2Dd (MHC Class I H-2Dd)

Alternative Name: MHC Class I H-2 Dd (H-2Dd Products)

Background: Synonyms: D-D alpha chain, H-2 class I histocompatibility antigen, H-2D(D), H2-D1

Gene ID: 100045864

NCBI Accession: XP_003086970

UniProt: P01900