Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids

Author(s)

da Silva, Marcelo Santos ^[1] ; Marin Munoz, Paula Andrea ^[1] ; Armelin, Hugo Aguirre ^[1] ; Elias, Maria Carolina ^[1]

Affiliation

^[1] Butantan Inst, Lab Especial Ciclo Celular, Ctr Toxins Immune Response & Cell Signaling CeTIC, 1500 Vital Brasil Ave, BR-05503900 Sao Paulo - Brazil

• Document type: Journal article
• Source: Journal of Eukaryotic Microbiology; v. 64, n. 6, p. 756-770, NOV-DEC 2017.
• Web of Science Citations: 10

Abstract

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and click chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re-estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring. (AU)

• FAPESP's process: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling
• Grantee: Hugo Aguirre Armelin
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 15/10580-0 - Characterization of intra-S checkpoint in Trypanosoma cells
• Grantee: Maria Carolina Quartim Barbosa Elias Sabbaga
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/24170-5 - DNA replication dynamics in Trypanosoma cruzi: licensing and replication rate characterization
• Grantee: Marcelo Santos da Silva
• Support Opportunities: Scholarships in Brazil - Post-Doctoral