Assessment of Different Permeabilization Methods of Minimizing Damage To the Adherent Cells For Detection of Intracellular Rna By Flow Cytometry

Background: various fixation and permeabilization techniques have been developed for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly rna. we tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18s rrna in hela cells. methods: hela cells were fixed in 2% paraformaldehyde. a variety of detergents and enzymes including saponin, tritonx-100, tween-20, np40, proteinase k, and streptolysin o were used to optimize a protocol of permeabilization for the flow cytometric enumeration of intracellular 18s rrna. treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of fitc-labeled sense and antisense probes to detect 18s ribosomal rnas. samples were then analyzed on a facscalibur flow cytometer. to evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with dapi, and evaluated on a fluorescent microscope with appropriate filter sets. results: in comparison with other methods, maximum cell frequency in percentage and fluorescent intensity (m1=2.1%, m2=97.9%) were obtained when the cells were treated with 0.2% tween-20 and incubated for 30 min (p= 0.001). conclusion: our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with tween-20. however, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized.