We engineered clones that stably express the light-emitting enzyme luciferase for the cell lines used, denoted Caov-3luc+, OV-90luc+, SKOV-3luc+and COV318luc+. In order to achieve stable expression of luciferase, we used the construct L192 for the OV-90luc+, Caov-3luc+ and SKOV-3luc+cell lines and the RediFect Red-FLuc-GFP lentiviral particles (cat # CLS960003, Perkin Elmer, Inc., Waltham, MA, USA) for COV318 both coding for the firefly luciferase enzyme. Infectious viral particles were harvested after 24 h of incubation. Retroviral infection was performed as described earlier [39] (link) and according to the manufactures protocol.
Redifect red fluc gfp lentiviral particles
Manufactured by PerkinElmer
Sourced in United States
RediFect Red-FLuc-GFP lentiviral particles are a tool for gene delivery. They contain a firefly luciferase (FLuc) reporter gene and a green fluorescent protein (GFP) reporter gene, both under the control of a constitutive promoter. These lentiviral particles can be used to transduce target cells and enable the detection of transduced cells through luciferase activity and GFP expression.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Cov318, by Merck Group (1 mentions)
Caov 3, by Merck Group (1 mentions)
Mycoalert plus assay, by Lonza (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
Skov3, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Skov3, by American Type Culture Collection (1 mentions)
Caov3, by American Type Culture Collection (1 mentions)
Pcdna3.1 backbone, by Thermo Fisher Scientific (1 mentions)
Facsaria fusion, by BD (1 mentions)
Facsaria 3 flow cytometric cell sorter, by BD (1 mentions)
Sbc 3, by PerkinElmer (1 mentions)
Redifect red fluc puromycin, by PerkinElmer (1 mentions)
Sbc 3, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
7 protocols using redifect red fluc gfp lentiviral particles
1
Engineered Luciferase-Expressing Ovarian Cancer Cell Lines
2
Establishing EOC Cell Lines for In Vitro and In Vivo Studies
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Human EOC cell lines Caov-3 (cat# HTB-75, RRID: CVCL_0201), OV-90 (cat# CRL-11732, RRID: CVCL_3768), and Skov-3 (cat# HTB-77, RRID: CVCL_0532) were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA), and COV318 (cat# 07071903, RRID: CVCL_2419) from Sigma Aldrich (Sigma Aldrich, St. Louis, USA). The cell lines were cultured in RPMI 1640 (OV-90) and DMEM (Caov-3, Skov-3 and COV318) media supplemented with 10% heat-inactivated FBS and 1% L -glutamine for at least one week (3–7 passages) before included into in vitro or in vivo studies (RPMI cat# R5886, DMEM cat# D5671, Sigma Aldrich) (FBS cat# 10270106, L -Glutamine cat# 25030081, Gibco, Paisley, UK). Mycoplasma testing was performed using the MycoAlert™ PLUS assay (cat# LT07-705, Lonza, Walkersville, USA). All cell lines were transduced to express green fluorescent protein (GFP) and red-shifted Luciola Italica luciferase; performed according to the manufacturers protocol using the RediFect Red-FLuc-GFP lentiviral particles (cat# CLS960003, Perkin Elmer, Waltham, MA, USA). Stable expression of luciferase allowed for non-invasive in vivo monitoring of tumour growth by bioluminescence imaging.
3
Generating Engineered Target Cell Lines
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To generate exogenous target cell lines, 96-well plates were seeded with COS-7 cells, and Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) was used to transfect the cells with indicated combinations of plasmids in the pcDNA3.1 backbone (Thermo Fisher Scientific, Waltham, MA). Cells were washed 24 h after transfection prior to use as target cells for T-cell co-culture assays. To generate a luciferase-expressing RPMI-6666 cell line, cells were transduced with RediFect Red-Fluc-GFP lentiviral particles (PerkinElmer, Waltham, MA). Transduced cells were sorted for GFP expression with a FACSAria Fusion (BD Biosciences, San Jose, CA) to obtain a purely transduced population. Engineered target cell lines were authenticated by short-tandem repeat (STR) profiling, and parental origin (100% exact match of 8 core STR loci) was confirmed by ATCC Cell Line Authentication Service (Manassas, VA).
4
Establishment of Luciferase- and GFP-expressing Cell Lines
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Luciferase- and GFP-expressing A431, MDAMB468,
3T3/Her2, and Calu3 cell lines were established by transducing them
with RediFect Red-FLuc-GFP lentiviral particles (PerkinElmer, Waltham,
MA).9 (link),10 (link),22 (link),23 (link) Their high luciferase expression was confirmed through
10 passages. Cells were cultured in RPMI 1640 medium (Thermo Fisher
Scientific Inc.) supplemented with 10% fetal bovine serum and 100
IU mL–1 penicillin/100 μg mL–1 streptomycin (Thermo Fisher Scientific Inc.).
3T3/Her2, and Calu3 cell lines were established by transducing them
with RediFect Red-FLuc-GFP lentiviral particles (PerkinElmer, Waltham,
MA).9 (link),10 (link),22 (link),23 (link) Their high luciferase expression was confirmed through
10 passages. Cells were cultured in RPMI 1640 medium (Thermo Fisher
Scientific Inc.) supplemented with 10% fetal bovine serum and 100
IU mL–1 penicillin/100 μg mL–1 streptomycin (Thermo Fisher Scientific Inc.).
5
Bioluminescent 4T1BR5 Cell Line
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Brain-seeking mouse mammary carcinoma cells (4T1BR5) were a kind gift from Dr. Patricia Steeg's lab (NIH, Centre for Cancer Research) and engineered to stably coexpress red-shifted Luciola italica luciferase (FLuc) and GFP using a commercial lentiviral vector (RediFect Red-FLuc-GFP lentiviral particles; PerkinElmer, USA). Cells were transduced at a multiplicity of infection of 20 and sorted based on GFP expression using a FACSAria III flow cytometric cell sorter (BD Biosciences, San Jose, CA, USA). The resultant 4T1BR5-FLuc-GFP cells were maintained in DMEM containing 10% FBS at 37°C and 5% CO2. All in vitro experiments were performed in triplicate.
6
Establishment of MCF7 Cell Lines with ERα Mutations
7
Chemoresistant Small Cell Lung Cancer Cell Lines
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The human SCLC cell line SBC‐3 was purchased from the Japanese Collection of Research Bioresources (JCRB). The cell line is authenticated by the agency. The chemoresistant cell lines of SBC‐3, SBC‐3/CDDP, SBC‐3/ETP (etoposide), and SBC‐3/SN‐38 (7‐ethyl‐10‐hydroxycamptothecin) were kindly provided by Dr. Katsuyuki Kiura (Okayama University, Okayama, Japan).21 , 22 , 23 The luciferase‐expressing cell lines (SBC‐3‐luc and SBC‐3/CDDP‐luc) were established by transduction with RediFect Red‐FLuc‐Puromycin or RediFect Red‐FLuc‐GFP lentiviral particles (PerkinElmer), as described previously.24 , 25 High luciferase expression was confirmed with more than 10 passages. RPMI‐1640 medium (Thermo Fisher Scientific) supplemented with 10% heat‐inactivated FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific) at 37°C under 5% CO2 atmosphere were used for the cell line cultures.
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