MHC Class II I-Ek Mouse Monoclonal Antibody [Clone ID: 14-4-4S]

Specifications

Product Data
Clone Name	14-4-4S
Applications	CT
Recommend Dilution	Cytotoxicity analysis.
Reactivity	Mouse
Host	Mouse
Clonality	Monoclonal
Immunogen	C3H Donor: C3H.SW Fusion Partner: SP2/0-Ag 14
Specificity	This monoclonal antibody is specific for cells expressing the Ia antigen coded for by the Ea subregion. The reaction pattern of this antibody with a panel of standard and recombinant haplotypes demonstrates that this antibody reacts with antigen Ia.m7, which is expressed by all haplotypes except b,f,q and s. This antibody is well suited for the quantitation and elimination of Ia bearing cells.
Formulation	PBS and 0.02% NaN3 State: Purified State: Liquid purified IgG
Concentration	lot specific
Purification	Protein G 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.
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 1:1000 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: CBA Cell Concentration: 1.1x10e6 cells/ml Complement: Low-Tox®-M Rabbit Complement Complement Concentration: 1:10 Procedure: Two-stage cytotoxicity Tissue Distribution by Cytotoxicity Analysis: Antibody Concentration Used: 1:500 Strain: CBA Cell Source - C.I. Thymus: 0 Spleen: 48 Lymph Node: 25 Bone Marrow: 16 Enriched B-cells: 65 Enriched splenic B-cells prepared by treatment of Lympholyte®-M purified spleen lymphocytes with Monoclonal Anti-Thy 1.2 Antibody plus complement, followed by removal of dead cells on lympholyte®-M. Strain Distribution by Cytotoxicity Analysis: Procedure: As above Antibody Concentration Used: 1:80 H-2 Loci Alleles: see FIGURE 2 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 1:500 and mix. Alternatively, pellet the cells and resuspend in antibody diluted 1:500 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: 1/25 Complement: Low-Tox®-M Rabbit Complement Complement Concentration Used: 1/8 Treatment of C3H/He and BALB/c splenocytes with this Ab plus complement resulted in a significant reduction in the number of plaque-forming cells. As assessed by a CTL assay, there was no effect on cytotoxic T cell function in both presensitized and postsensitized treated samples. 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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