MHC Class II I-Ak Mouse Monoclonal Antibody [Clone ID: 14V.18]

Specifications

Product Data
Clone Name	14V.18
Applications	CT, FN
Recommend Dilution	Cytotoxicity Analysis. Functional assay.
Reactivity	Mouse
Host	Mouse
Clonality	Monoclonal
Immunogen	A.TL Donor: A.TH Fusion Partner: P3-X63-Ag8
Specificity	This monoclonal antibody is a cytotoxic antibody specific for cells expressing the Ia antigen coded for by the A subregion of the k haplotype. The reaction pattern of this antibody with a panel of inbred and recombinant haplotypes demonstrates that the antibody reacts with Ia.m2, a private specificity of the H-2k haplotype. This antibody can be used to quantitate or to eliminate cells bearing the I-Ak (Ia.m2) antigen.
Formulation	PBS containing 0.02% Sodium Azide as preserative. State: Purified State: Liquid purified Ig fraction.
Concentration	lot specific
Purification	Protein A Chromatography.
Conjugation	Unconjugated
Storage Condition	Store the antibody undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer. Avoid repeated freezing and thawing.
Database Link	UniProt ID P01910
Synonyms	H2-Aa
Note	Protocol: CYTOTOXICITY ANALYSIS: Method: 1. Prepare a cell suspension from the appropriate tissue in Cytotoxicity Mediuma or equivalent. Remove red cells and dead cells (where necessary) by purification of viable lymphocytes on Lympholyte®-Mb 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 1:500 - 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 Low-Tox®-M Rabbit Complementc diluted to the recommended concentration in Cytotoxicity Medium.. 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 hemacytometer. Cytotoxic Index (C. I.) can be calculated as shown in Figure 1. Results - Antibody Titration by Cytotoxicity Analysis: Cell Source: Enriched Splenic B Cells Donor: C3H/He Cell Concentration: 1.1x10e6 cells/ml Complement: Low-Tox®-M Rabbit Complement Complement Concentration: 1:12 Procedure: Two-stage cytotoxicity SEE FIGURE 2 Results - Tissue Distribution by Cytotoxicity Analysis: Antibody Concentration Used: 1:40 Strain: C3H/He Cell Source C.I. Thymus: 4 Spleen: 41 Lymph Node: 17 Bone Marrow: 4 Enriched B-cells: 90 Results - Strain Distribution by Cytotoxicity Analysis: SEE FIGURE 3 Antibody Concentration Used: 1:40 CYTOTOXICITY DEPLETION ASSAY: Method: 1. Prepare a cell suspension from the appropriate tissue in Cytotoxicity Mediuma or equivalent. Remove red cells and dead cells (where necessary) by purification of viable lymphocytes on Lympholyte®-M 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 Low-Tox-M® Rabbit Complementc, diluted to the appropriate concentration in Cytotoxicity Medium. (Recommended concentration included with 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 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 hemacytometer. 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 cell suspension over a 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. Alternatively, 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. FUNCTIONAL TESTING: Method: Cells were treated as described in Cytotoxicity Depletion Assay. Treated cells and controls were tested for: a) the ability to generate plaque-forming cells (PFC) using a modified Jerne haemolytic plaque assay. b) the ability to generate cytotoxic T effector cells using a cytotoxic lymphocyte reaction (CTL) assay. Cells were treated both before and after sensitization in the CTL assay. Results: Cell Source: Splenocytes Donors: C3H/He and BALB/c Cell Concentration: 1x10e7 cells/ml Antibody Concentration Used: 1:200 Complement: Low-Tox®-M Rabbit Complement Complement Concentration Used: 1:10 Treatment of C3H/He splenocytes with this Ab plus complement resulted in a significant reduction in the number of plaque-forming cells. Partial inhibition of cytotoxic T effector cell function as assessed by CTL assay was also noted. Treatment of BALB/c cells had no effect on either plaque-forming cell number or cytotoxic T cell function. These results are consistent with the removal of I-Ak bearing cells and their related activities. NOTES: a. Cytotoxicity Medium is RPMI-1640 with 25 mM 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. 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. b. Lympholyte®-M cell separation medium is a 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. c. 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. Low-Tox®-M 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.
Reference Data

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