Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry

ABSTRACT

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. 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. 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]. 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