How can I increase the transduction efficiency with the BacMam 2.0 reagents such as the the CellLight and Premo products?
Try varying particle-to-cell ratio (PPC), incubation volume, temperature and, cell density (if adherent cells are transduced). For adherent cells, we recommend a confluence of about 70%. Following the PPC, adjusting the volume is the next best parameter to change to optimize protein expression. If that doesn't work, you can also use the BacMam Enhancer Kit (Cat. No. B10107).
'We present an integrated method that uses extended time-lapse automated imaging to quantify the dynamics of cell proliferation. Cell counts are fit with a quiescence-growth model that estimates rates of cell division, entry into quiescence and death. The model is constrained with rates extracted experimentally from the behavior of tracked ... More
Analysis of nucleocytoplasmic transport using green fluorescent protein.
Authors:Stauber RH,
Journal:Methods Mol Biol
PubMed ID:12136753
A hallmark of eukaryotic cells is their spatial and functional separation into the nucleus and the cytoplasm by the nuclear envelope. Although this separation introduces a potent and sophisticated level of regulation, it also requires highly effective and selective transport machinery. ... More
Functional Screening Assays with Neurons Generated from Pluripotent Stem Cell-Derived Neural Stem Cells.
Authors:Efthymiou A, Shaltouki A, Steiner JP, Jha B, Heman-Ackah SM, Swistowski A, Zeng X, Rao MS, Malik N,
Journal:
PubMed ID:24019252
Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for ... More
Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleus.
Authors:Roth DM, Harper I, Pouton CW, Jans DA,
Journal:J Cell Biochem
PubMed ID:19507231
Nuclear protein transport processes have largely been studied using in vitro semi-intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the ... More
Assessment of GFP expression and viability using the tali image-based cytometer.
Authors:Remple K, Stone L,
Journal:J Vis Exp
PubMed ID:22127256
Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward ... More