Simultaneous flow cytometric measurement of K-562 megakaryocytic differentiation and CD56+ large granular lymphocyte cytotoxicity.
K-562 cells have the capacity to undergo multi-lineage differentiation, which may be crucial to their ability to serve as target reservoirs for CD56+ large granular lymphocytes (LGL). Conventional techniques using chromium release assays to measure lymphocyte-mediated cytotoxicity suffer from disadvantages, including radioactive contamination and the inability to simultaneously determine K-562 and/or CD56+ lymphocyte phenotypes. We illustrate here a three-color flow cytometric method providing for the simultaneous evaluation of K-562-CD56+ LGL binding, K-562 cell viability, and the status of K-562 cell differentiation. Phorbol 12-myristate 13-acetate (PMA) engenders megakaryocytic differentiation in K-562 cell populations, as measured by presentation of the beta(3) integrin (gpIIIa, CD61), while maintaining a negative expression of MHC-I and MHC-II molecules. Using the auto-fluorescence of K-562 cells, flow cytometry can be used to demonstrate a significant decrease in CD56+ LGL activity against K-562 cells in populations pre-incubated with PMA. The capacity of three-color flow cytometry to measure lymphocyte-target cell binding and cell death kinetics, while simultaneously determining target cell phenotype, permits the specific localization of CD61-expressing K-562 cells to areas inconsistent with CD56+ LGL-mediated patterns of lysis.
Baker
E J
EJ
Department of Medical Biology, Graduate School of Medicine, University of Tennessee Medical Center at Knoxville, 1924 Alcoa Highway, 37920, Knoxville, TN, USA.
Ichiki
A T
AT
Day
N E
NE
Andrews
R B
RB
Bamberger
E G
EG
Lozzio
C B
CB
D015703:Antigens, CD / Q000032:analysis; D019002:CD56 Antigen / Q000032:analysis*; D016923:Cell Death; D002454:Cell Differentiation; D003116:Color; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D039661:Integrin beta3; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism; D007700:Kinetics; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000276:immunology*; D008533:Megakaryocytes / Q000276:immunology*; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015703:Antigens, CD; D019002:CD56 Antigen; D039661:Integrin beta3; D010980:Platelet Membrane Glycoproteins; D013755:Tetradecanoylphorbol Acetate