A long-term flow cytometry assay to analyze the role of specific genes of Drosophila melanogaster S2 cells in surviving genotoxic stress.
Drosophila S2 cells are easy to manipulate and culture and are a versatile model system for high-throughput screens such as genome-wide siRNA screens to find genes involved in stress or therapy resistance or for screening through large compound libraries to identify cytotoxins. Clonogenic assays are considered the gold-standard to investigate the cytotoxicity of specific treatments or to compare the sensitivity of various cell types for a specific treatment. However, this assay cannot be used for Drosophila S2 cells as they are virtually unable to grow in distinct colonies. We designed a novel fluorescence-based flow cytometry assay to study long-term proliferation of S2 cells under various conditions and in the presence of specific gene products or after downregulation of specific gene products. Here we validate this assay and we used this novel method to investigate the role of checkpoint genes grapes/Dchk1 and DmChk2 in cell survival responses. Our data demonstrate that Grapes/Dchk1 but not DmChk2 is required to survive hydroxyurea. Our assay will be of use to investigate the long-term effects of various treatments in S2 cells and to evaluate the role of specific proteins therein.
Yi
Xia
X
Department of Radiation and Stress Cell Biology, Division of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Lemstra
Willy
W
Vos
Michel J
MJ
Shang
Yongfeng
Y
Kampinga
Harm H
HH
Su
Tin Tin
TT
Sibon
Ody C M
OC
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D002469:Cell Separation; D002470:Cell Survival; D004249:DNA Damage; D029721:Drosophila Proteins / Q000737:chemistry; D004331:Drosophila melanogaster; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000737:chemistry; D009153:Mutagens; D034622:RNA Interference
D029721:Drosophila Proteins; D009153:Mutagens; D049452:Green Fluorescent Proteins