Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines their primary effector function is through target cell lysis. within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562 721.221 and Jurkat we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay inside the same experimental set up. Image cytometry can accurately analyze live focus on cells by excluding dimmer cells and smaller sized apoptotic physiques from viable focus on cell matters. The picture cytometry-based cytotoxicity assay can be a simple immediate and sensitive technique and can be an interesting option for regular cytotoxicity assay. Intro Organic killer (NK) cells are innate immune system cells that become the first type of protection against tumor cells and different pathogens . The effector features of NK cells consist of immune rules through secretion of cytokines such as for example interferon-γ and TNF-α by a subset (Compact disc56bcorrect Compact disc16?) . Nevertheless the major mode of actions from the main A 922500 subset of NK cells (Compact disc56dimCD16+) is the direct lysis of their targets . Therefore assessment of NK cell cytolytic function is fundamental to the study of NK cell biology and application in adoptive immunotherapy. The cytolytic activity of Rabbit Polyclonal to GFP tag. NK cells is assessed either through a degranulation assay (LAMP1/CD107a)  or through a cytotoxicity assay. The degranulation assay although very useful in assessing percentage of NK cells that respond to a stimuli (such as a tumor target) it does not provide any information about the outcome of the response such as cytolysis of the tumor targets following the degranulation assault by NK cells. Therefore cytotoxicity assays are important in the context of understanding the cytolytic impact of NK cells and to measure the sensitivity of a given tumor target for lysis by NK cells. Cytotoxicity assays are thus more commonly used to assess the functional efficacy of NK cells for adoptive immunotherapy applications. Several assays have been developed for determining cytotoxicity of immune cells; use of 14Chromium was first reported in 1964  and the 51Chromium release assay (CRA) was described in 1968 . To date CRA is considered the ‘gold standard’ for measuring NK cell and cytolytic T cell cytotoxicity [7-11]. However due to concerns over the toxicity of handling and disposing radioactive compounds several methods have been developed as alternatives to CRA. One alternative method based on a non-toxic fluorescent dye using Calcein AM (acetoxymethyl) was developed in 1994 . Other methods include flow-based cytotoxicity assays [13-17] LDH release assays [18-20] and more recently a bioluminescence-based method . Some of these methods A 922500 show good correlation of target cell lysis to CRA [17 22 23 while others show greater target cell lysis than CRA [13 21 The calcein release assay was demonstrated by Neri S. et al. to possess good relationship to CRA at evaluating percent particular lysis . Therefore we have regularly utilized the calcein launch assay for confirming NK cell cytotoxicity inside our A 922500 research [24 25 Nevertheless we have noticed that calcein includes a divergent launching efficiency in various cell lines and calcein offers been proven to possess A 922500 higher spontaneous launch in comparison to 51Chromium (51Cr) . Large spontaneous launch and lower launching efficiency in a few tumor cell lines may lead to decreased powerful range and reduced level of sensitivity from the assay. Additionally mainly because the calcein launch assays measure focus on cell lysis from the launch of entrapped calcein in to the supernatant an imperfect launch of calcein from lysed cells A 922500 could result in underestimation of the percent lysis of target cells. Target cell death following interaction with immune effectors such as NK cells and cytotoxic T lymphocytes is.