MHC Class II I-Abd Mouse Monoclonal Antibody [Clone ID: 28-16-8S]

CAT#: CL067P

MHC Class II I-Abd mouse monoclonal antibody, clone 28-16-8S, Purified

Product Images

Figure 1: Cytotoxic Index

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  Figure 3

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  Figure 2

Specifications

Product Data

• Clone Name: 28-16-8S
• Applications: CT
• Recommended Dilution: Flow Cytometry.
  Can be used to quantitate or eliminate cells bearing the I-Ab antigen from the appropriate strains of mice.
• Reactivities: Mouse
• Host: Mouse
• Isotype: IgM
• Clonality: Monoclonal
• Immunogen: C3H.SW spleen
  Donor: C3H
  Fusion Partner: SP2/0Ag.14
• Specificity: This monoclonal antibody reacts with the I-Ab encoded MHC class II antigen expressed on mouse strains of the H-2b haplotype. It also reacts with the I-Ad encoded MHC class II antigen expressed on mouse strains of the H-2d haplotype. The reaction pattern of this antibody with a panel of inbred and recombinant haplotypes demonstrates that the antibody detects the public Ia.m8 determinant. Class II antigens are most highly expressed on antigen-presenting cells including B cells, macrophages, dendritic cells and certain epithelial cells.
• Formulation: PBS and contains 0.02% sodium azide (NaN3) as a preservative.
  State: Purified
  State: Liquid purified IgM
• Concentration: lot specific
• Purification: Affinity chromatography
• Conjugation: Unconjugated
• Storage: Store the antibody undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer.
  Avoid repeated freezing and thawing.
• Stability: Shelf life: one year from despatch.
• 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 1.1x10e6 cells per ml in Cytotoxicity Medium.
  2. Add the antibody to a final concentration of 1:640 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:
  see FIGURE 2
  Cell Source: Splenocytes
  Donor: BALB/c
  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: 1:640
  Strain: BALB/c
  
  Cell Source - C.I.
  Thymus: 0
  Spleen: 61
  Lymph Node: 31
  Bone Marrow: 17
  
  Strain Distribution by Cytotoxicity Analysis:
  see FIGURE 3
  Procedure: As above
  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. Afterwashing, adjust the cell concentration to 1x10e7 cells per ml in Cytotoxicity Medium.
  2. Add the antibody to a final concentration of 1:640 and mix. Alternatively, pellet the cells and resuspend in antibody diluted 1:640 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.
  
  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.