title: Efficacy of Local Anesthetics in Detachment of Normal 3T3 Mouse Fibroblasts and Prostate Cancer AT-2 Cells from Substrata, in Maintenance of Viable Cells in a Non-Adherent State, and in Preservation of Cell Surface Markers Detected with FlowSight Image Cytometry.
abstract: The local anesthetics procaine, lidocaine and tetracaine permit the reversible detachment of viable cells and their passaging or preservation in a non-adherent state in the absence of proteolytic enzymes. The effects of these anesthetics, dissolved in various media, on cell viability, cell detachment from substrata and preservation of cells in a non-adherent state, were compared using the AT-2 line of rat prostate carcinoma cells of moderate malignancy and the 3T3 mouse fibroblast cell line. It was found that all three local anesthetics can induce cell rounding followed by detachment of over 95% of viable cells in both lines in Ca2+/Mg(2+)-free PBS. Tetracaine in 1 mM concentration was the most effective in induction of fast cell detachment. However, procaine and lidocaine in 16 mM concentrations were found to be optimal for preservation of cells in a non-adherent state and for the maintenance of cell viability for at least 2 h. The tested anesthetics also cause cell rounding and detachment when present in various cell culture media but these processes occurred much more slowly and less efficiently than in Ca2+/Mg(2+)-free PBS. Normal 3T3 mouse fibroblasts after detachment and passaging undertake growth reaching the same saturation density in cultures after detachment with procaine or lidocaine as after passaging using trypsin solution. The results suggest that the application of local anesthetics can be a very simple and effective technique for cell passaging in tissue cultures. This technique might decrease side-effects and cell injury caused by trypsinization or cell scraping. The preservation of cells in suspension in a non-adherent state may facilitate analysis of cell surface properties and fractionation of cell mixtures. Avoiding the use of trypsin allows for the preservation of cell surface proteins ICAM, CXCR4, and HCAM analyzed with FlowSight image flow cytometry.
authors.forename: Włodzimierz
authors.initials: W
authors.lastname: Korohoda
authors.forename: Jarosław
authors.initials: J
authors.lastname: Oczkowicz
authors.forename: Kamil
authors.initials: K
authors.lastname: Rolski
authors.forename: Damian
authors.initials: D
authors.lastname: Ryszawy
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Madeja
mesh_terms: D016475:3T3 Cells; D000779:Anesthetics, Local / Q000494:pharmacology*; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D005434:Flow Cytometry / Q000295:instrumentation*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011471:Prostatic Neoplasms; D051381:Rats
chemical_list: D000779:Anesthetics, Local; D008565:Membrane Proteins