Detection of S-Phase of Cell Division Cycle in Plant Cells and Tissues by Using 5-Ethynyl-2′-Deoxyuridine (EdU)

Abstract

Assaying cell proliferation is crucial in assessing cell health, characterizing cellular responses to various treatments or genetic modifications. It is also essential to determine the degree of synchronicity in cell division cycle synchronization experiments. Most cell proliferation assays estimate the number of cells by either incorporating a modified nucleotide during cell proliferation or by measuring the total nucleic acid content of lysed or isolated cells. The most accurate method is direct measurement of new DNA synthesis, which traditionally was achieved by the incorporation of tritium-labeled thymidine and detection by autoradiography [1, 2]. This radioactive method has been replaced by others, such as the incorporation of the thymidine analog, 5-bromo-2-deoxyuridine (BrdU), into DNA followed by immunodetection with a specific antibody raised against BrdU [3, 4]. Although effective and sensitive, this method requires DNA denaturation or digestion (using hydrochloric acid, heat, or DNase) to expose BrdU to the antibody [5, 6]. The use of acid or heat often destroys cell morphology and damages the epitope of many proteins. This hinders their immunocytochemical detection with fluorescence-labeled antibodies. Using DNase, however, poses difficulty in obtaining reproducible levels of DNA digestion while analyzing different cell types or samples [7].The antibody-based detection method of BrdU assay also necessitates cell wall digestion in experiments carried out on plant cells. Therefore, protoplasts or partially cell wall-digested cells/organs/tissue sections are often used for BrdU-based detection of proliferative activity in plants [8]. However, a significant wounding and osmotic stress is imposed on live plant cells due to treatment with cell walldigesting enzymes. Moreover, specific optimization of the digestion medium, of the type and concentration of the enzymes, and their osmolarity is required for

Conformational Changes and Nuclear Entry of Porcine Circovirus without Disassembly

“…Once the labeled genome is released, it is visible by a click reaction with fluorophore azide (28). Nucleoside analogues have been widely used for DNA labeling and for cell and organism metabolism studies (29,30). Compatible with immunocytochemistry, DNA metabolic labeling with "clickable" nucleoside analogues and copper (I)-catalyzed azide-alkyne cycloaddition reactions allow DNA visualization without sample denaturation.…”