Testing Data
I-Ed (private; Ia.m23) Monoclonal Antibody
Anti-Mouse I-Ed (private; Ia.m23), Ascites (Clone 34-1-45) (mouse IgM)
Synonyms
I-Ed (private; Ia.m23); Monoclonal Antibody; Anti-Mouse I-Ed (private; Ascites (Clone 34-1-45) (mouse IgM); Anti-Mouse I-Ed Monoclonal Antibody-Ascites; anti-I-Ed (private; Ia.m23) antibody
Clonality
Monoclonal
Isotype
Mouse IgM
Clone Number
34-1-4S
Specificity
Anti-I-Ed (private determinant Ia.m23)
Form/Format
0.5 ml, lyophilized ascites
Application Notes
RECOMMENDED METHOD FOR DEPLETING A CELL POPULATION OF I-Ed BEARING LYMPHOCYTES:
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® denstiy cell concentration to 1 x 107 cells per ml in cytotoxicity medium.
2. Add the antibody to a final concentration of 1:20 and mix. Alternatively, pellet the cells and resuspend in antibody diluted 1:20 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, resuspend to the original volume in Low-Tox-M® Rabbit Complement diluted 1/10 in cytotoxicity medium. If using splenocytes or bone marrow cells, resuspend to the original volume in Baby Rabbit Complement 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 form 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 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 I-Ed 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 cells concentration to 1 x 106 cells per ml in cytotoxicity medium.
2. Add the antibody to a final concentration of 1:40 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, resuspend to the original volume in Low-Tox—M® rabbit complement diluted 1:10 in cytotoxicity medium. If using splenocytes or bone marrow cells, resuspend 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 hemaytometer. Cytotoxic index (C.I) can be calculated as follows:
C.I. + % cyt. (antibody + complement)/100% - % cyt. (complement alone)/- % cyt. (complement alone)
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® denstiy cell concentration to 1 x 107 cells per ml in cytotoxicity medium.
2. Add the antibody to a final concentration of 1:20 and mix. Alternatively, pellet the cells and resuspend in antibody diluted 1:20 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, resuspend to the original volume in Low-Tox-M® Rabbit Complement diluted 1/10 in cytotoxicity medium. If using splenocytes or bone marrow cells, resuspend to the original volume in Baby Rabbit Complement 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 form 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 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 I-Ed 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 cells concentration to 1 x 106 cells per ml in cytotoxicity medium.
2. Add the antibody to a final concentration of 1:40 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, resuspend to the original volume in Low-Tox—M® rabbit complement diluted 1:10 in cytotoxicity medium. If using splenocytes or bone marrow cells, resuspend 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 hemaytometer. Cytotoxic index (C.I) can be calculated as follows:
C.I. + % cyt. (antibody + complement)/100% - % cyt. (complement alone)/- % cyt. (complement alone)
Sterility
This reagent is not sold as sterile, but can be sterilized by filtration if necessary. To minimize loss of volume during filtration, dilute to the final working concentration in the appropriate medium before filtration and filter through a 0.45um filter
Immunization
Recipient: C3H
Donor: B6xDBA/2
Donor: B6xDBA/2
Presentation
Ascites Fluid; 0.45um filtered
Strain Distribution
I-Ed positive strains
Note
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 I 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 wil 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.
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 wil 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.
Preparation and Storage
Store at -20°C or below before reconstitution. Reconstitute with 0.5ml of distilled water. Aliquot and freeze the unused portion in volumes appropriate for single usage (as repeated freezing and thawing may cause loss of antibody activity).
Testing Data
Testing Data
Testing Data
Testing Data
Related Product Information for anti-I-Ed (private; Ia.m23) antibody
anti-mouse I-Ed monoclonal antibody detects the private specificity of the I-Ed antigen (determinant Ia.m23).
Similar Products
Product Notes
The I-Ed (private; Ia.m23) (Catalog #AAA520369) is an Antibody and is intended for research purposes only. The product is available for immediate purchase. RECOMMENDED METHOD FOR DEPLETING A CELL POPULATION OF I-Ed BEARING LYMPHOCYTES: 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® denstiy cell concentration to 1 x 107 cells per ml in cytotoxicity medium. 2. Add the antibody to a final concentration of 1:20 and mix. Alternatively, pellet the cells and resuspend in antibody diluted 1:20 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, resuspend to the original volume in Low-Tox-M® Rabbit Complement diluted 1/10 in cytotoxicity medium. If using splenocytes or bone marrow cells, resuspend to the original volume in Baby Rabbit Complement 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 form 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 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 I-Ed 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 cells concentration to 1 x 106 cells per ml in cytotoxicity medium. 2. Add the antibody to a final concentration of 1:40 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, resuspend to the original volume in Low-Tox—M® rabbit complement diluted 1:10 in cytotoxicity medium. If using splenocytes or bone marrow cells, resuspend 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 hemaytometer. Cytotoxic index (C.I) can be calculated as follows: C.I. + % cyt. (antibody + complement)/100% - % cyt. (complement alone)/- % cyt. (complement alone). Researchers should empirically determine the suitability of the I-Ed (private; Ia.m23) for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "I-Ed (private; Ia.m23), Monoclonal Antibody" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
If you are ready to order, navigate to Shopping Cart and get ready to checkout.
