Analysis of in vitro lymphocyte adhesion and transendothelial migration by fluorescent-beads-based flow cytometric cell counting.
In this report, we describe a new and simple method for flow cytometric quantitation of lymphocyte numbers in lymphocyte-endothelial adhesion/ transendothelial migration assays. The method exploits fluorescent flow cytometer alignment beads as a counting reference. Known amounts of beads are added to samples with unknown lymphocyte numbers. Lymphocytes and a preset number of fluorescent beads are simultaneously analyzed. The total number of cells present in the sample can be subsequently calculated from the fixed ratio of added to analyzed fluorescent beads. Using this fluorescent-beads-based flow cytometric cell counting of lymphocyte numbers in adhesion/migration assays, labeling of cells and other time-consuming calibration procedures are not required and analysis time is short. Furthermore, we demonstrate that this cell counting method can be combined with concurrent single- or double-label fluorescence flow cytometric phenotyping of adherent and migrated lymphocytes. The method was applied to the in vitro study of the effects of lymphocyte activation status and binding of bispecific antibody (directed against CD3 x tumor cell-associated antigen) on lymphocyte adhesion and transendothelial migration.
Molema
G
G
Department of Clinical Immunology, Groningen Utrecht Institute for Drug Exploration, Groningen, The Netherlands.
Mesander
G
G
Kroesen
B J
BJ
Helfrich
W
W
Meijer
D K
DK
de Leij
L F
LF
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000032:analysis; D017252:CD3 Complex / Q000032:analysis; D002448:Cell Adhesion / Q000276:immunology*; D015815:Cell Adhesion Molecules / Q000032:analysis; D002452:Cell Count / Q000379:methods; D002465:Cell Movement / Q000276:immunology*; D002469:Cell Separation; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000276:immunology*; D000071858:Epithelial Cell Adhesion Molecule; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000166:cytology*; D008863:Microspheres; D010641:Phenotype; D015203:Reproducibility of Results; D014471:Umbilical Veins
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D017252:CD3 Complex; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D007140:Immunoglobulin Fab Fragments