(a) Schematic of permeabilization, delivery, for reversible permeabilization using streptolysin O (SLO) on live CHO-K1 cells. Cells are exposed to SLO for 7–10 min before incubating with the three different fluorescent probes (DAPI, phalloidin-Alexa647, and anti-PMP70-ATTO488) on ice for 5 min. Recovery was initiated by adding 10% FBS DMEM supplemented with nucleotide ATP, GTP, and glucose. (b) Specific labeling of cellular structures by SLO delivered probes. Nucleus, peroxisome, and actin filaments are labeled by DAPI, Anti-PMP70-ATTO488, and phalloidin-Alexa647 respectively. The cells are then incubated with Texas red- 20 kDa dextran to check whether the membrane has recovered. The negative control (SLO-) shows no labeling of cellular structure. The negative control cells are then permeabilized by SLO to show the fluorescence of Texas Red in permeabilized cells. (DAPI is impermeant to CHO-K1 cells.) (c) p65 translocating from cytosol to nucleus after SLO permeabilization in HeLa cells. TNF-α and Leptomycin B (LMB) were used to stimulate nucleus import of the p65-GFP protein labeled by GBP-ATTO647N. The nucleus fluorescence of both GFP and ATTO647N channels increased 15 and 30 min after stimulation as shown in time series fluorescence images (Left). The percent increase of nucleus fluorescence was quantified (Right). (Red square = ATTO647N intensity in the nuclear after TNF-α and LMB treatment, Green circle = GFP intensity after TNF-α treatment [N = 4], Red circle =ATTO647N Intensity after TNF-α treatment [N = 4]. Blue circle with dashed line is the GFP intensity after TNF-α treatment for cells that have never been permeabilized by SLO [N = 4]. Green triangle = GFP intensity without TNF-α and LMB treatments [N = 3]). Scale bar denotes 10 μm.