MHC Class I H-2 Kb / H-2 Db Mouse Monoclonal Antibody [Clone ID: 5041.16.1]

Specifications

Product Data
Clone Name	5041.16.1
Applications	CT
Recommend Dilution	Cytotoxicity assay (see protocol).
Reactivity	Mouse
Host	Mouse
Clonality	Monoclonal
Specificity	This antibody antibody is specific for cells expressing the H-2K antigen coded for by the b haplotype and for cells expressing the H-2D antigen coded for by the b haplotype.
Formulation	PBS + 0.02 % NaN3 State: Purified State: Liquid Ig fraction
Concentration	lot specific
Purification	Protein G chromatography
Conjugation	Unconjugated
Storage Condition	Store the antibody at 2 - 8 °C up to one month or (in aliquots) at -20 °C for longer. Avoid repeated freezing and thawing.
Synonyms	HLA Class I
Note	Protocol: RECOMMENDED METHOD FOR DEPLETING A CELL POPULATION OF H- 2Kb AND/OR H-2Db BEARING LYMPHOCYTES: 1. Prepare a cell suspension from the appropriate tissue in Cytotoxicity Medium1 or equivalent. Remove red cells and dead cells (where necessary) by purification of viable lymphocytes on density cell separation medium. After washing, adjust the cell concentration to 1x10e7 cells per ml in Cytotoxicity Medium. 2. Add the antibody to a final concentration of 1:100 and mix. Alternatively, pellet the cells and resuspend in antibody diluted 1:100 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 Rabbit Complement, diluted to the appropriate concentration in Cytotoxicity Medium. (Recommended concentration included with each batch of Rabbit Complement.) 6. Incubate for 60 minutes at 37°C. 7. Monitor for percent cytotoxicity at this stage, before further processing. For this purpose remove a small sample from each tube, dilute 1:10 with medium, and add 1/10 volume of 1% 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 further processing, particularly if the treated cells are to be cultured. This can be done by layering the treated cell suspensions over an equal volume of cell suspension separation medium and centrifuging at room temperature as per the instructions provided. Live cells will form a layer at the interface, while the dead cells pellet. The interface can then be collected and washed in Cytotoxicity Medium before being resuspended in the appropriate medium for further processing. Alternately, the cells can be washed and resuspended in the appropriate medium for further processing immediately after Step 6., provided that the dead cells will not interfere with subsequent assays. RECOMMENDED METHOD FOR DETERMINING PERCENT OF H-2Kb AND/ OR H-2Db BEARING CELLS IN A POPULATION: 1. Prepare a cell suspension from the appropriate tissue in Cytotoxicity Medium 1 or equivalent. Remove red cells and dead cells (where necessary) by purification of viable lymphocytes on density cell separation medium. After washing, adjust the cell concentration to 1x10e6 cells per ml in Cytotoxicity Medium. 2. Add the antibody to a final concentration of 1:1000 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 Rabbit Complement3 diluted to the appropriate concentration in Cytotoxicity Medium. (Recommended concentration included with each batch of 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 before scoring works well. Score live versus dead cells in a haemacytometer. Cytotoxic Index (C.I.) can be calculated as follows: C.I. = 100 x %cyt. (antibody + complement) - % cyt. (complement alone)/ 100 - % cyt. (complement alone) NOTES: 1. Cytotoxicity Medium is RPMI-1640 with 25mM Hepes buffer and 0.3% bovine serum albumin (BSA). BSA is substituted for the conventionally used fetal calf serum (FCS) because we have found that many batches of FCS contain complement dependent cytotoxins to mouse lymphocytes, thus increasing the background killing in the presence of complement. We recommend that cells not be exposed to FCS prior to or during exposure to antibody and complement. Some batches of BSA also contain complement dependent cytotoxins, resulting in the same problem. We screen for batches of BSA giving low background in the presence of complement and use the selected BSA for preparing Cytotoxicity Medium. 2. Cell separation medium is density separation medium designed specifically for the isolation of viable mouse lymphocytes. This separation medium provides a high and non-selective recovery of viable mouse lymphocytes, removing red cells and dead cells. The density of this medium is 1.087-1.088. Isolation of mouse lymphocytes on cell separation medium of density 1.077 will result in high and selective loss of lymphocytes and should be avoided. 3. Rabbit serum provides the most potent source of complement for use with antibodies to mouse cell surface antigens. However, rabbit serum itself is very toxic to mouse lymphocytes. Rabbit Complement is absorbed to remove toxicity to mouse lymphocytes, while maintaining its high complement activity. When used in conjunction with Cytotoxicity Medium, this reagent provides a highly potent source of complement with minimal background toxicity. ANTIBODY TITRATION: Cell Source: Splenocytes Donors: C57BL/6 Cell Concentration: 1x10e6 cells per ml Complement: Rabbit Complement Complement Concentration: 1:10 Procedure: Two stage cytotoxicity as described on page 3, "Recommended Method for Determining Percent of H-2Kb or H-2Db bearing cells in a Population". C.I. = Cytotoxic Index = 100 x %cyt. (antibody + complement) - % cyt. (complement alone)/ 100 - % cyt. (complement alone) (see picture below) STRAIN DISTRIBUTION: Procedure: As above Antibody Concentration: Final concentration 1:100 Strains Tested: C3H/He, C57BL/6, B10-A(4R), BIO-A(5R), B6Lyt 2.1 3.1, C57BL/10 SnJ, CBA/J, AKR/J, BALB/c Cells Killed by Treatment: C57BL/6, BIO-A(4R), BIO-A(5R),
Reference Data

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