Fluorescence Microscopy Imaging:
REF52 cells were plated in a chambered coverglass and transduced with lentiviral particles at an MOI of 20 for 24 hours. After media replacement and 48 hours further incubation, cells were imaged live by oil immersion wide-field fluorescence microscopy. The EB3-GFP displays a punctate distribution characteristic of microtubule ends. Immunocytochemistry Comparison and Inhibitor Analysis:
HeLa cells were plated in a coverglass chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. EB3-GFP displays a nearly complete overlap with EB3 detected by immunocytochemical staining with rabbit polyclonal anti-EB3 (EMD Millipore cat. no. AB6033). Additional Cell Types:
MCF7 cells were transduced with EB3-RFP lentivirus as in Figure 1 at an MOI 40 for 24 hours, then stained for tubulin. EB3-GFP displays a punctate and short linear distribtion. Immunocytochemical staining of the same field of view with a monoclonal antibody against α-tubulin (EMD Millipore Cat. No. 05-829) reveals to the typical filamentous pattern of microtubules. Overlay of EB3-GFP (green), anti-tubulin (red) and DAPI (blue) demonstrates that EB3-GFP is localized to the microtubule ends. Hard-to-transfect Cell Types:
Primary cell type HUVEC and Human mesenchymal stem cells (HuMSC) were plated in coverglass chamber slides and transduced with EB3-GFP lentiviral particles at an MOI of 20 and 40, respectively, for 24 hours. Time-lapse Imaging:
REF52 cells were plated in coverglass chamber slides and transduced with EB3-GFP lentiviral particles. Images were collected every 3 seconds for a total of 2 min. Shown here are 3 sequential frames; to see the entire sequence, please visit our Cell Structure-Biosensors
page. For optimal fluorescent visualization, it is recommended to analyze the target expression level within 24-48 hrs after transfection/infection for optimal live cell analysis, as fluorescent intensity may dim over time, especially in difficult-to-transfect cell lines. Infected cells may be frozen down after successful transfection/infection and thawed in culture to retain positive fluorescent expression beyond 24-48 hrs. Length and intensity of fluorescent expression varies between cell lines. Higher MOIs may be required for difficult-to-transfect cell lines.