Incorporation of fluorescein-conjugated anti-mouse immunoglobulin G into permeabilized Nicotiana tabacum BY-2 cells

A simple and efficient procedure for the transient permeabilization of cult ured cells has been developed for the incorporation of a high molecular wei ght substance, fluorescein isothiocyanate-conjugated anti-mouse immunoglobu lin G (FITC-IgG, 150 kDa) into Nicotiana tabacum BY-2 cells grown in suspen sion cultures. The cells were treated with cell permeabilization solutions (CPS) comprising varying concentrations of glycerol (Gly), sucrose (Suc), e thylene glycol (EG) and dimethyl sulfoxide (DMSO). When the cells were perm eabilized by treating with CPS-3 (Gly:Suc:EG:DMSO = 20:5:20:5, w/v%) contai ning FITC-IgG for 15 s at room temperature followed by washing with ice-col d 1 M sucrose and modified LS medium, 76% of the cells incorporated FITC-Ig G and 78% of the cells remained Viable according to fluorescein diacetate ( FDA) staining. Most treated cells retained intact internal structure and th e incorporated FITC-IgG was detected in cytoplasm and in the nuclear region . Cells of different ages were studied. Four day-old cells were found suita ble for the incorporation of FITC-IgG and cell survival. Inclusion of 10 mM CaCl2 in CPS-3 improved both FITC-IgG uptake and cell survival. The regrow th of treated cultures lagged for the first 3 days, then followed a growth pattern similar to the untreated controls. (C) 1999 Elsevier Science Irelan d Ltd. All rights reserved.