I-Ed (Private; Ia.m23) Antibody – Monoclonal

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)}