CHAPTER ELEVEN Granzymes and Cell Death Denis Martinvalet,* Jerome Thiery,* and Dipanjan Chowdhury † Contents 1. Introduction 214 2. Purification of Rat Perforin from RNK-16 NK-like Leukemia Cells 215 2.1. Growth of RNK-16 cells in rats 215 2.2. Cytotoxic granules isolation from RNK-16 cells 216 2.3. Purification of rat perforin 217 2.4. Perforin activity detection 219 3. Expression and Purification of Recombinant Granzyme B in a Baculovirus System 221 3.1. Material/reagent/solution 222 3.2. Growth of Sf9 and H5 cells 222 3.3. Virus production 222 3.4. Virus amplification 222 3.5. GzmB expression using H5 cells 223 3.6. GzmB purification 223 3.7. GzmB activity assay 223 4. Expression and Purification of Recombinant human Granzyme A in Escherichia coli 223 4.1. Material/reagent/solution 224 4.2. Solutions 224 4.3. Expression of GzmA in E. coli 224 4.4. Purification of GzmA protein 225 4.5. Measuring GzmA activity 226 5. Granzyme-Mediated Apoptosis Assay 227 5.1. Perforin titration 227 5.2. Granzyme A- and B-mediated cell death assay 227 6. Conclusion 229 Acknowledgment 229 References 229 Methods in Enzymology, Volume 442 # 2008 Elsevier Inc. ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)01411-0 All rights reserved. * Immune Disease Institute and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts { Dana Farber Cancer Institute and Department of Radiation Oncology, Harvard Medical School, Boston, Massachusetts 213