Figure 2. Resealing of semi-intact HeLa cells by the addition of CaCl₂.

A. Basic protocol for the resealing of semi-intact HeLa cells with CaCl₂. B. HeLa cells were permeabilized with SLO, and were incubated with 1.5 mg/ml L5178Y cytosol, an ATP regenerating system, GTP, glucose, and fluorescein-conjugated dextran (ATP/GTP/glucose/fluorescein-dextran) at 32°C for 15 min. The cells were incubated for a further 5 min at 32°C with or without 1 mM CaCl₂. Resealed (CaCl₂-treated) or semi-intact (CaCl₂-untreated) cells were incubated with DMEM supplemented with 10% FCS or TB at 37°C for 30 min. The cells were observed under a confocal microscope. Staining with both fluorescein-dextran (green) and PI (red) indicated that the cells had been resealed efficiently. Semi-intact cells were positive for PI staining but not for fluorescein-dextran. Bar  =  20 µm. C. Semi-intact HeLa cells were incubated with 0.0, 1.5, 3.0, 5.0, or 7.0 mg/ml L5178Y cytosol and ATP/GTP/glucose/fluorescein-dextran at 32°C for 15 min, and then were resealed as described in A. Means and standard deviations for the percentage of resealed cells, which were stained with both PI and fluorescein-dextran, are shown in the graph. D. Semi-intact HeLa cells were incubated with L5178Y cytosol as described in A, and then were resealed by treatment with 0.0, 0.1, 0.5, 1.0, or 3.0 mM CaCl₂ at 32°C for 5 min. Means and standard deviations for the percentage of PI- and fluorescein-dextran-positive resealed cells are shown in the graph. E. Semi-intact HeLa cells were incubated with L5178Y cytosol as described in A, and then were resealed by treatment with 1 mM CaCl₂ at 32°C for 0.0, 1.5, 3.0, 5.0, or 7.5 min. Means and standard deviations for the percentage of PI- and fluorescein-dextran-positive resealed cells are shown in the graph. F. Semi-intact HeLa cells were incubated with 1.5 mg/ml L5178Y cytosol and ATP/GTP/glucose/fluorescein-dextran at 32°C for 15, 30, 60, or 90 min, and then were resealed as described in A. Means and standard deviations for the percentage of PI- and fluorescein-dextran-positive resealed cells are shown in the graph.