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 cultured cells has been developed for the incorporation of a high molecular weight substance, fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (FITC-IgG, 150 kDa) into Nicotiana tabacum BY-2 cells grown in suspension cultures. The cells were treated with cell permeabilization solutions (CPS) comprising varying concentrations of glycerol (Gly), sucrose (Suc), ethylene glycol (EG) and dimethyl sulfoxide (DMSO). When the cells were permeabilized by treating with CPS-3 (Gly:Suc:EG:DMSO=20:5:20:5, w/v%) containing FITC-IgG for 15 s at room temperature followed by washing with ice-cold 1 M sucrose and modified LS medium, 76% of the cells incorporated FITC-IgG and 78% of the cells remained viable according to fluorescein diacetate (FDA) staining. Most treated cells retained intact internal structure and the incorporated FITC-IgG was detected in cytoplasm and in the nuclear region. Cells of different ages were studied. Four day-old cells were found suitable for the incorporation of FITC-IgG and cell survival. Inclusion of 10 mM CaCl 2 in CPS-3 improved both FITC-IgG uptake and cell survival. The regrowth of treated cultures lagged for the first 3 days, then followed a growth pattern similar to the untreated controls.