Cell Culture -- Chromium Release Assay



In cellular immunology, the mechanisms in which the body wards off foreign substances such as viruses, bacteria and toxic agents is a major area of investigation. The key player in the body's defense system against these toxic invaders is the cytotoxic T-lymphocyte. These lymphocytes have receptors which recognize antigens present on the surface of foreign cells. Recognition of a foreign antigen by the receptor allows the cytotoxic T-lymphocyte to proceed to kill the invading cells.

In vitro methods, known as Chromium Release Assays, have been developed which allow researchers to quantify this cytotoxic phenomenon. Among other things, these assays allow one to determine the number of lymphocytes being produced in response to an infection or a drug treatment and how effectively these cells kill the foreign antigen bearing cells.

The basis of the assay is chromium 51 which has the property of binding to the cellular proteins of cultured cells. Target cells are pre-labeled by incubation with 51Cr. These target cells are then incubated with effector cells and the amount of radioactivity which is released in the supernatant is taken as an indicator of the amount of lysis which has occurred. This assay takes advantage of the fact that cytotoxic T-cells kill their target cells by disrupting the integrity of the cell membrane thereby allowing the release of 51Cr bound to protein from the target cell. A simple calculation of the amount of cell bound 51Cr versus free 51Cr allows one to quantify the amount of cellular cytotoxicity.

Procedure

1. Cultured cells are given a dose of radiolabeled sodium chromate 51Cr. As the cells grow, they uptake the 51Cr into cellular proteins. At the end of the incubation, the cells are washed to remove any unincorporated label.

2. Effector (E) cells are incubated with chromium labeled target (T) cells at various cell ratios (denoted as E/T ratios). Incubation is typically performed in a multiwell plate for a period of 2-4 hours at 37C. Cytotoxic effects mediated by the effector cells are quantitated by measuring the released 51Cr-protein complex. After incubation, the multiwell plate is centrifuged to pellet the cells to the bottom of the plate. An aliquot is removed from each of the wells and quantitated using a gamma counter. The rest of the well's contents (cells and supernatant) is removed using one of several commercially available cell harvesters which then transfers the material to glass fiber filters. Each of the filters is counted in a gamma counter.

3. The percent cytotoxicity/cell lysis is calculated based on the total amount of 51Cr released. In short, the calculation is as follows: