The use of the CELLection kit in the isolation of carcinoma cells from mononuclear cell suspensions.
A study was performed to evaluate in vitro the sensitivity, specificity and variability of a new immunomagnetic microbead isolation technique which provides subsequent immunological staining of captured carcinoma cells. In a mixture of peripheral blood mononuclear cells (PBMCs) and human carcinoma cells the epithelial cancer cells were isolated with the Dynal((R)) RAM IgG1 CELLection Kit using Dynabeads M-280 coated with a rat monoclonal antibody (Mab) against mouse IgG1. The rat Mab was biotinylated and attached to Dynabeads via streptavidin and a DNA linker. The anti-epithelial monoclonal mouse antibody Ber-EP4 was used as the primary capture antibody. In order to permit phenotyping of the isolated carcinoma cells the magnetic beads were removed from the carcinoma cells by DN'ase digestion of the DNA linker between the magnetic bead and the secondary antibody. In an ex vivo model system an average recovery of approximately 60% of a human colon carcinoma cell line HCC-2998 seeded in 5.10(6) PBMCs was obtained, and the recovered cells could subsequently be immunologically stained for the surface antigen CD87 (urokinase plasminogen activator receptor). No positive stained cells were found in control experiments with PBMCs without carcinoma cells. Despite a relatively low recovery, the described method will be valuable for the detection of carcinoma cells in cytospin preparations with subsequent phenotyping of the cells for expression of surface antigens. Depending on the chosen antibodies, the method may be useful for the isolation and characterisation of other cell types in various cell suspensions.
Werther
K
K
Department of Surgical Gastroenterology 235, Hvidovre Hospital, University of Copenhagen, 2650, Hvidovre, Denmark. k.werther@forum.dk
Normark
M
M
Hansen
B F
BF
Brünner
N
N
Nielsen
H J
HJ
D000818:Animals; D002277:Carcinoma / Q000473:pathology*; D002469:Cell Separation / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D051379:Mice; D051381:Rats; D011933:Reagent Kits, Diagnostic; D013194:Staining and Labeling / Q000379:methods; D014407:Tumor Cells, Cultured
D011933:Reagent Kits, Diagnostic