title: Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids.
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.
authors.affiliation: Laboratório Especial de Ciclo Celular, Center of Toxins, Immune Response and Cell Signaling (CeTICS), Butantan Institute, 1500, Vital Brasil Avenue, 05503-900, São Paulo, Brazil.
authors.forename: Marcelo Santos
authors.initials: MS
authors.lastname: da Silva
authors.affiliation: Laboratório Especial de Ciclo Celular, Center of Toxins, Immune Response and Cell Signaling (CeTICS), Butantan Institute, 1500, Vital Brasil Avenue, 05503-900, São Paulo, Brazil.
authors.forename: Paula Andrea Marin
authors.initials: PAM
authors.lastname: Muñoz
authors.affiliation: Laboratório Especial de Ciclo Celular, Center of Toxins, Immune Response and Cell Signaling (CeTICS), Butantan Institute, 1500, Vital Brasil Avenue, 05503-900, São Paulo, Brazil.
authors.forename: Hugo Aguirre
authors.initials: HA
authors.lastname: Armelin
authors.affiliation: Laboratório Especial de Ciclo Celular, Center of Toxins, Immune Response and Cell Signaling (CeTICS), Butantan Institute, 1500, Vital Brasil Avenue, 05503-900, São Paulo, Brazil.
authors.forename: Maria Carolina
authors.identifier: 0000-0002-0501-8280
authors.initials: MC
authors.lastname: Elias
mesh_terms: D001973:Bromodeoxyuridine / Q000032:analysis*; D002453:Cell Cycle; D004261:DNA Replication; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000032:analysis; D007891:Leishmania / Q000254:growth & development* / Q000378:metabolism; D013194:Staining and Labeling; D014345:Trypanosoma / Q000254:growth & development* / Q000378:metabolism
chemical_list: D001973:Bromodeoxyuridine; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine
keywords: 
  Leishmania amazonensis
  ; 
  Trypanosoma brucei
  ; 
  Trypanosoma cruzi
  ; Cytokinesis; mitosis