Single cell assay with an automated capillary microinjection system.
An automated capillary microinjection system with computer-controlled positioning of the cells and of the capillary, and its applications and advantages, is described. The system is easy in handling and manipulation. About 1500 injections are possible in 1 h, with high reproducibility. In cytoplasmic and nuclear injections more than 90 and 85% of the cells are successfully injected. Using FITC-dextran at a concentration of 0.5% as a fluorescently labeled coinjection marker, 99% of the cells can be retrieved in culture medium even 48 h after injection. The coordinates of the cells are stored in the computer and accuracy in statistical evaluation of experiments is improved in comparison to the manual techniques. Methods for preparation and handling of glass capillaries were developed resulting in reproducible form and significantly reduced clogging rate. The improved characteristics offered by this system are demonstrated in studies leading to the confirmation of existence of an mRNA inhibiting DNA synthesis in cells. Functional screening by cell injections of cDNA libraries and of size-fractionated mRNA molecules can be performed efficiently with the automated microinjection system.
Pepperkok
R
R
European Molecular Biology, Laboratory, Heidelberg, Federal Republic of Germany.
Schneider
C
C
Philipson
L
L
Ansorge
W
W
D001345:Autoradiography; D001562:Benzimidazoles; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D003911:Dextrans / Q000008:administration & dosage; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins / Q000008:administration & dosage; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D008099:Liver; D008113:Liver Neoplasms; D008838:Microcomputers; D008845:Microinjections / Q000379:methods*; D008846:Micromanipulation; D012333:RNA, Messenger / Q000008:administration & dosage; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured
D001562:Benzimidazoles; D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D012333:RNA, Messenger; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate; C017807:bisbenzimide ethoxide trihydrochloride