Author/Authors :
Amidzadeh، Zahra نويسنده Diagnostic Laboratory Sciences and Technology Research Center, Shiraz University of Medical SciencesStudent Research Committee, Shiraz University of Medical Sciences , , Behzad-Behbahani، Abbas نويسنده , , Erfani، Nasrollah نويسنده Cancer Immunology Group, Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran , , Sharifzadeh، Sedigheh نويسنده Diagnostic Laboratory Sciences and Technology Research Center, Shiraz University of Medical Sciences , , Ranjbaran، Ali Reza نويسنده Division of Uro-oncology, Department of Urology, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran , , Moezzi، Leili نويسنده Diagnostic Laboratory Sciences and Technology Research Center, Shiraz University of Medical Sciences , , Aboualizadeh، Farzaneh نويسنده , , Okhovat، Mohammad Ali نويسنده Student Research Committee, Shiraz University of Medical Sciences, Shiraz, IR Iran , , Alavi، Parniyan نويسنده Diagnostic Laboratory Sciences and Technology Research Center, Shiraz University of Medical Sciences , , Azarpira، Negar نويسنده ,
Abstract :
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.
