Isolation of cytotoxic T cell and NK granules and purification of their effector proteins.
Killer lymphocytes induce apoptosis by the release of cytotoxic mediators from specialized secretory lysosomes, called cytotoxic granules, into the immunological synapse formed with a cell targeted for elimination. Methods are presented here for isolating CTL and NK cell cytotoxic granules using cell disruption by nitrogen cavitation followed by continuous Percoll density gradient fractionation. Protocols are also given for purifying the key cytolytic molecules (perforin, granzyme A, granzyme B, and granulysin) from isolated cytotoxic granules by fast protein liquid chromatography.
Program in Cellular and Molecular Medicine, Children's Hospital and Immune Disease Institute, Boston, Massachusetts, USA.
Jerome
J
Thiery
Michael
M
Walch
Danielle K
DK
Jensen
Denis
D
Martinvalet
Judy
J
Lieberman
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D003594:Cytoplasmic Granules / Q000737:chemistry*; D006801:Humans; D007694:Killer Cells, Natural / Q000737:chemistry*; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D013602:T-Lymphocytes, Cytotoxic / Q000737:chemistry*
D011506:Proteins