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Cytotoxicity: ⁵¹Chromium Release

What this assay tells you

Effector Cytotoxic T Lymphocytes (CTL) bind targets (infected cells) bearing virus peptide on syngeneic (self) Class I MHC and signal the targets to undergo apoptosis. If the targets are labeled with ⁵¹Chromium before the CTL are added, the amount of ⁵¹Cr released into the supernatant is proportional to the number of targets killed.

What you need to do the assay

• Cell suspension containing CD8 T cells which have been activated with peptide plus self Class I MHC in vivo. It is not necessary to purify CD8 T cells, but it is helpful to remove granulocytes and monocyte-macrophages.
• Tissue culture supplies.
• ⁵¹Cr

How the assay is done

• Incubate the infected cells with ⁵¹Cr; wash. Cells spontaneously take ⁵¹Cr into their cytosol.
• Incubate CTL with ⁵¹Cr-labeled target cells for 4-8 hours. As target cells die, they release ⁵¹Cr into the culture supernatant.
• Centrifuge the cells and count the amount of ⁵¹Cr in the supernatant (specific release).
• Negative controls eliminate the CTL and measure spontaneous release of ⁵¹Cr from the target cells. Positive controls use detergent to lyse target cells and determine maximum ⁵¹Cr release.
• Percent specific cytotoxicity is calculated by

  (specific release - spontaneous release) ÷ (maximum release - spontaneous release)

  and may be 20%-85% for a positive assay. Percent specific cytotoxicity is usually determined at several ratios of Effector (CTL) to target cells (E:T).

How to interpret the results

The higher the amount of ⁵¹Cr released into the culture supernatant (percent specific cytotoxicity), the more targets have been killed by the CTL.

Additional comments

The frequency of specific CTL is very low in a primary response and CTL are difficult to activate in vitro. For these reasons, the ⁵¹Cr release assay is usually done with cells stimulated in vivo. Although CTL can each kill many targets in vivo, in vitro this assay requires numbers of CTL equal to or greater than the number of targets for detectable killing.

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