Conditions for permeabilization of cells used for intracellular tyrosine phosphorylation studies.

Permeabilized cells are widely applied for studying the effect of membrane impermeant biomolecules on signaling pathways. After permeabilization with reagents commonly used for this purpose, the architecture of the cells remains essentially intact, and hence, they retain their ability to respond to cell surface receptor stimulation as far as early cellular responses are concerned. Early signaling events, such as tyrosine and serine phosphorylation of cellular proteins (3,6), generation of cAMP (1) or inositol-phosphate hydrolysis (3) can be investigated after the stimulation of permeabilized cells. Several procedures have been reported to produce permeable cells. These methods apply bacterial toxins such as tetanolysin (1,2,9) and streptolysin-O (SLO) (2,3) or detergents like digitonin (5) and L-α-lysophosphatidyl choline (LPC) (6,8) as permeabilizing agents. However, a common feature of most of the permeabilizing reagents is that they induce a significant tyrosine phosphorylation in most of the leukemic cell lines, which might obscure the regulatory effect of the biomolecules that are tested (References 6 and 9, and unpublished observation). Our aim was to establish a permeabilization procedure that resolves this problem. Therefore, we first compared different reagents, namely SLO, digitonin and LPC and various conditions for permeabilization to select the most efficient system. SLO-mediated permeabilization required a high concentration of SLO (200 IU/mL) and a long time (30 min) to fully permeabilize 107 J6 cells or peripheral blood mononuclear cells (PBMC) at 37°C (data not shown). Permeabilization with digitonin also required 10–30 min, and it was not well reproducible. The LPCmediated permeabilization has turned out to be the most reliable, because it was highly reproducible in the case of different cells (leukemic cell lines, peripheral mononuclear cells), and the permeabilization was completed in a short time. Therefore, the LPC-mediated permeabilization (6) was characterized and optimized for studying the effect of various biomolecules on tyrosine phosphorylation. The amount of LPC, the appropriate time and the optimal temperature required for permeabilization have been established in a series of experiments. The LPC of 50 μg/mL permeabilized a wide variety of cell lines at 107–108 cell/mL concentration within 1 min (data not shown). Permeabilization carried out at 4° or 37°C was equally efficient (data not shown). Permeability of the cells was assessed in each experiment using trypan blue exclusion. The efficiency of the LPC permeabilization was also assessed by introducing fluorescein isothiocyanate (FITC)-labeled peptides of 2 and 4 kDa molecular mass (data not shown) and FITC-labeled immunoglobulin (150 kDa) into the cells (Figure 1). An antibody recognizing the intracellular domain of the CD43 molecule stained over 95% of the permeabilized cells confirming the successful permeabilization (Figure 1). The method we finally preferred for permeabilization was as follows: the human leukemic T cells (J6) were centrifuged, washed once with the cell culture medium RPMI (Life Technologies,