Article Title: Non-Invasive Fluorescent Monitoring of Ovarian Cancer in an Immunocompetent Mouse Model
Figure Lengend Snippet: iRFP720 fluorescence and characterisation of pROSA-iRFP720-expressing ID8 cells. ( A ) Fluorescence-activated cell sorting (FACS) enrichment of iRFP720-expressing ID8 cells. iRFP720-positive single cells ([R]730/45 vs. [R]670/30) were sorted into a 96-well plate using the FACSAria Fusion cell sorter (BD Biosciences, San Jose, CA, USA), and single clones were expanded and screened. ( B ) iRFP720 fluorescence was observed using the Cytation 3 imaging Multi-Mode Reader equipped with a Cy5.5 filterset (BioTek Instruments Inc., Winooski, VT, USA). Hoechst 33342 was used to visualise the nucleus (377 ex /447 em ). ( C ) Schematic diagram representing the genomic screening strategy used for identifying iRFP720 incorporation into the ROSA26 locus. ( D ) Genomic DNA isolated from pROSA-puro-iRFP720-transfected single-cell ID8 clones was PCR-amplified using a ROSA26 upstream F primer, an endogenous ROSA26 -specific R primer and an iRFP720-specific R primer, and PCR products were separated with a 1.2% agarose gel. Wild-type ROSA26 gives an 869bp product and successful iRFP720 incorporation gives a 2883bp product. C1–5: clones 1–5; WT: wild-type ID8; NTC: no-template control. ( E ) Proliferation was assessed by electrode impedance using xCELLigence real-time cell analysis. Cells (8 × 10 3 /well) were treated with cisplatin (10 µg/mL) or paclitaxel (20 nM) after 8 h, and proliferation was analysed for further 64 h. ( F ) Wild-type and pROSA-iRFP720 ID8 (3 × 10 5 cells/well) were cultured for 24 h, and then incubated for further 24 h in the presence of cisplatin (10 µg/mL) or paclitaxel (20 nM). Apoptosis was determined by Alexa Fluor®647 annexin V and PI staining on the BD LSRFortessa X-20 (BD Biosciences) flow cytometer. ( G ) Representative images of the apoptosis analysis. Data are presented as mean ± SD, n = 3.
Article Snippet: Transfected ID8 cells were maintained in puromycin selection for two weeks and then sorted by flow cytometry for single iRFP720+ clones ([R]730/45 vs. [R]670/30) using the FACSAria Fusion cell sorter (BD Biosciences, San Jose, CA, USA).
Techniques: Fluorescence, Expressing, FACS, Clone Assay, Imaging, Isolation, Transfection, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Cell Culture, Incubation, Staining, Flow Cytometry, Cytometry