MHC Class I H-2Ld Mouse Monoclonal Antibody [Clone ID: 30-5-7S]

Specifications

Product Data
Clone Name	30-5-7S
Applications	CT
Recommend Dilution	Cytotoxicity Analysis (See Protocol).
Reactivity	Human
Host	Mouse
Clonality	Monoclonal
Immunogen	Recipient: BALB/c-H-2dm2 Donor: BALB/c Spleen cells Fusion Partner: SP2/0.Ag14
Specificity	This Monoclonal antibody detects the public specificity H-2.65 of the H-2Ld antigen. This antibody also recognizes H-2Dq and H-2Lq molecules.
Formulation	PBS State: Aff - Purified State: Liquid purified IgG fraction Preservative: 0.02% Sodium Azide
Purification	Affinity Chromatography on Protein G
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 P01897
Synonyms	H2-L
Note	Protocol: RECOMMENDED METHOD FOR DEPLETING A CELL POPULATION OF H-2Ld BEARING LYMPHOCYTES: 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 Lympholyte-M®2 cell separation medium. After washing, adjust cell concentration to 1 x 107 cells per ml in cytotoxicity medium. 2. Add the antibody to a final concentration of 1:25 and mix. Alternatively, pellet the cells and resuspend in antibody diluted 1:25 in cytotoxicity medium. 3. Incubate for 60 minutes at 4°C. 4. Centrifuge to pellet the cells and discard the supernatant. 5. If using thymocytes or lymph node cells, it is recommended that the cells be resuspended to the original volume in Low-Tox-M® Rabbit Complement3 diluted 1:10 in cytotoxicity medium. If using splenocytes or bone marrow cells, it is recommended that the cells be resuspended to the original volume in Baby Rabbit Complement 4 diluted 1:10 in cytotoxicity medium. 6. Incubate for 60 minutes at 37°C. 7. If desired, 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 vs. 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 an equal volume of Lympholyte-M® cell 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 will 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. RECOMMENDED METHOD FOR DETERMINING PERCENT OF H-2Ld BEARING CELLS IN A POPULATION: 1. Prepare a cell suspension from the appropriate tissue in Cytotoxicity Medium® or equivalent. Remove red cells and dead cells (where necessary) by purification of viable lymphocytes on Lympholyte-M® density cell separation medium. After washing, adjust the cell concentration to 1 x 106 cells per ml in cytotoxicity medium. 2. Add the antibody to a final concentration of 1:50 and mix. 3. Incubate for 60 minutes at 4°C. 4. Centrifuge to pellet the cells and discard the supernatant. 5. If using thymocytes or lymph node cells, it is recommended that the cells be resuspended to the original volume in Low-Tox-M® Rabbit Complement diluted 1:10 in cytotoxicity medium. If using splenocytes or bone marrow cells, it is recommended that the cells be resuspended to the original volume in Baby Rabbit Complement diluted 1:10 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 vs. dead cells in a hemacytometer. The Cytotoxic Index (C.I) can be calculated as follows: C.I = 100x %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. Lympholyte-M® 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. 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. 4. Baby Rabbit Complement provides high complement activity with low background toxicity. ANTIBODY TITRATION: See Figure 1. Cell Source: Splenocytes Donors: BALB/c Cell Concentration: 1 x 106 cells/ml Complement: Low Tox®-M Rabbit Complement Complement Concentration: 1:10 Procedure: Two stage cytotoxicity Tissue Distribution: Antibody Concentration: 1:40 Strain: BALB/c Cell Source - C.I. Thymus: 55 Spleen: 95 Lymph Node: 98 Bone Marrow: 54 Strain Distribution: Antibody Concentration: 1:20 Strains Tested: Strain Haplotype +/- BALB/c H-2d + A.TH H-2KsDd + A.TL H-2KsDd + B.10A(3R) H-2KbDd + C3H/He H-2k - C57BL/6 H-2b - Functional Testing: Cell Source: Splenocytes Donors: BALB/c and C3H/He Cell Concentration: 1 x 107 cells/ml Antibody Concentration: 1:25 Complement: Baby Rabbit Complement Complement Concentration: 1:10 Procedure: Cells were treated as described in Recommended Method for Depleting a Cell Population of H-2Ld Bearing Lymphocytes. Treated cells and controls were tested for: a) the ability to generate plaque-forming cells (PFC) using a modified Jerne haemolytic plaque assay and 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. In vitro immunizations were used in all experiments. Results: Treatment of BALB/c splenocytes with anti-H-2Ld plus complement resulted in a marked reduction in the number of plaque-forming cells and essentially eliminated cytotoxic T cells, as assessed by Jerne haemolytic plaque assay and CTL assay, respectively. Treatment of C3H/He splenocytes had no effect. These resultra are consistent with the depletion of H-2Ld bearing cells.
Reference Data

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