title: [Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3].
abstract: OBJECTIVE
  To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3).
  
  
  METHODS
  A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment.
  
  
  RESULTS
  The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment.
  
  
  CONCLUSIONS
  The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.
authors.affiliation: Department of Medical Immunology and Laboratory, Medical School, Anhui University of Science and Technology, Huainan 232001, China.
authors.forename: Wan
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Medical Immunology and Laboratory, Medical School, Anhui University of Science and Technology, Huainan 232001, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Medical Immunology and Laboratory, Medical School, Anhui University of Science and Technology, Huainan 232001, China.
authors.forename: Runpeng
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Department of Medical Immunology and Laboratory, Medical School, Anhui University of Science and Technology, Huainan 232001, China.
authors.forename: Xuewei
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Clinical Laboratory, Affiliated Cancer Hospital, Anhui University of Science and Technology, Huainan 232001, China.
authors.forename: Yingru
authors.initials: Y
authors.lastname: Xing
authors.affiliation: Department of Medical Immunology and Laboratory, Medical School, Anhui University of Science and Technology, Huainan 232001, China.
authors.forename: Rongbo
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Medical Immunology and Laboratory, Medical School, Anhui University of Science and Technology, Huainan 232001, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Medical Immunology and Laboratory, Medical School, Anhui University of Science and Technology, Huainan 232001, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Hu
mesh_terms: D000818:Animals; D001343:Autophagy; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D000067996:RAW 264.7 Cells; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D000097573:Red Fluorescent Protein; C480860:Map1lc3b protein, mouse