...MagFRET: The First Genetically Encoded Fluorescent Mg(2+) Sensor. PLoS One. 2013 Dec 2;8(12):e82009. doi: ... there is one problem: how to get that into the cells of interest,” Griesbeck said. Because they are chemical...understand the connection between molecular- and cellular-level events and behavior. It’s a problem that...advantage for observing events taking place inside cells that are on the move, such as T lymphocytes. Griesbeck...-binding protein, troponin C (TnC), from muscle cells. They identified a toadfish TnC variant which had...cAMP-responsive element-binding protein (CREB) in living cells. “Now we are making this really good,” Griesbeck...FRET-based sensor proteins, particularly for the intracellular imaging of transition metal ions, such as zinc...

... & Walker S. The Bacterial Cell Envelope. Cold Spring Harb Perspect Biol. 2(5) a000414 (2010). Cleveland...negative bacteria is due to differences in cell wall composition. Cell wall structure Gram positive bacteria...complex within the peptidoglycan layer, staining the cell purple. When the alcohol is added, it dehydrates...

... and whether I would be able to carry out a yeast-2-hybrid library screen; instead I thought they would...would continue on with my academic dream of studying cell division. However, after 6 years in the business...Cambridge University where he studied mechanisms of cell division. Recently Seán got the entrepreneurial ...information on ELISA troubleshooting, ELISA kits and cell death assays. You can find Seán on twitter @Macfhearraigh...

...created for the Brunello library is shown in Figure 2. You can use a small PCR product because the plasmids... Typically, Addgene uses electrocompetent Stbl4 cells when performing amplifications, and we use electroporation...DNA. This bias can be counteracted by growing the cells on solid plates, where each transformant forms its...own colony, uninfluenced by the growth of other cells. For libraries where the plasmids are all very similar...

...project my team & I have poured our hearts into for 2 yrs) has 200K+ figures by 95K+ scientists 👨🏽‍🔬👩‍...four main categories: anatomy and the human body, cellular biology, medical specialities, and general items...

... master's degree prepared them for their career.  2. Are you looking for career advancement and growth... or so I thought. So I entered a molecular and cellular biology PhD program in 2011, which I left four...

...motion videos is very time consuming; producing a 1-2 minute video takes an entire week of our free time... to mold it all into a full laboratory. It is priceless to see our “toys” gaining life and working as ...

...much has been done with CRISPR technology in just 2 years and excitement about how much more there is ...alone were worth the trip. Dr. Dana Carroll's excellent keynote address was the first of 19 talks given...

...available to the community. We now offer AAV9 for 2 more constructs from Christopher Harvey’s lab. These... results, but also confirm expression in target cells. While the various AAV serotypes can be used to ...

...of translation 2-10 ug/mL G418/Geneticin neo HeLa, NIH3T3, CHO, 293HEK, Jurkat T cells Blocks polypeptide...transfected cells may be considered an entirely new cell line from that of the original parental cells. Positive...cultured mammalian cells. Of course, these mammalian vectors must be compatible with the cell type they are...bacterial cells); however, the techniques and reagents vary. Plasmid transfection into mammalian cells is fairly...expression levels in the cells. Ideally, the chosen reporter is unique to the cell, is expressed from the...components expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr...mammalian cells To achieve stable transfection, there should be a selective pressure to force cells to incorporate...

...differentiation.   Fig. 2: Single cell RNA-seq clustering of cells transduced with the TF pooled...human embryonic stem cells. Transduced stem cells were differentiated and single cell RNA-seq was performed...transduced human embryonic stem cells with the viral TF library, with each cell receiving only 1 TF expression...construct. Cells were cultured following a differentiation protocol before performing single-cell RNA sequencing... 206 cells per TF ORF. From this large sequencing endeavor, a map of possible TF-induced cellular states... clusters of individual cells along with color coded nominated reference cell types.  (Adapted from Joung...delivered to each cell, future applications could deliver multiple TFs to each cell, allowing for combinatorial...

...enrich the microvesicles containing AAVs (Figure 2). Since the development of the exo-AAV isolation protocol...the producer cells and purification protocols often describe a process that begins with cell lysis to release...particles is able to infect more cells (as demonstrated in different cellular environments such as retina ... steps to separate cell culture supernatant containing exo-AVVs from producer cells, and then to enrich...Exosomal vesicles secreted from producer cells also contain cellular macromolecules (e.g. proteins, nucleic...effectively target less accessible cells, such as brain tumor stromal cells in a mouse model of glioblastoma...Standard AAV vectors are generated with a producer cell line like the widely used 293T. During vector production...

...is the most charged, with 5 negatively charged and 2 positively charged amino acids. FLAG tag has been .... This tag is not suitable for use in apoptotic cells as Caspase3/7 both cleave after the DVPD sequence...fluorescent marker. The GFP tag is ideal for live cell imaging, FRET, flow cytometry, or immunofluorescence...

...yourself greater chances for success.  Build a team of 2 or 3 Don’t go it alone.  You’ll find that no matter...would rather fund excellent people with mediocre ideas than mediocre people with excellent ideas. I am lucky...

...incorporation of biosensors, protein evolution lineages, 2-photon spectra, more elaborate spectrum analysis tools...  There are many online guides – including an excellent series of posts by Joachim Goedhart on the Addgene...Associate at Harvard Medical School. He manages the Cell Biology Microscopy Facility, specializing in advanced...

...create a recombinant antibody.     Figure 2: Changes to the heavy chain or light chain genes within...isolating the B cells from an immunized animal, those B cells are fused to myeloma cells at a 1:1 ratio...fusion between an antibody-producing B cell and a myeloma cell (National Research Council (US) Committee...immortal cell population that will continue to produce the specific antibody that the original B cell produced... selects against unfused myeloma cells and antibody-producing B cells that cannot replicate, meaning that...monoclonal antibodies are derived from a single B cell clone. One common way of producing monoclonal antibodies...with BioRender.com.   The antibody-producing B cells that are used to make hybridomas come from animals...

...library was used to transduce T helper type 2 (Th2) cells from mice that constitutively express Cas9 and...genome-wide interrogation of the genes required for cellular processes. For a successful screen, it is necessary... on the method used to introduce the gRNAs into cells. Most CRISPR pooled libraries have been created ...the chance of transducing a only a single gRNA per cell. The viral DNA containing the gRNA then integrates...next-generation sequencing of the genomic DNA. However, some cell types are poorly transduced by lentivirus, and thus...CRISPR libraries was not possible. Primary murine T cells cannot be transduced by lentivirus and have been...CRISPR pooled library can be used for screening of cells that are refractory to lentiviral transduction, ...

... therapy.” Journal of cellular physiology.  https://doi.org/10.1002/jcp.30064 2. Barman et al. 2020. ...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (5 December...CRISPR in adult stem cells has huge potential, because the genomes of adult stem cells in organoid cultures...Genetic Disease in Mouse Via Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (5 December 2013). 8. Gerald Schwank...homologous recombination in cultured intestinal stem cells of patients with cystic fibrosis (8).   The difference...applied CRISPR to disease correction in adult stem cells isolated from two patients with cystic fibrosis....is a big advantage over induced pluripotent stem cells, which need to go through a phase of crisis where...

...improve the brightness of previous Nano-lanterns by 2-4 fold and permit five-color fluorescent imaging! ...have become indispensable tools for molecular and cell biologists over the last two decades, but their ...light. Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be...quantum yield, and when transiently expressed in cells, generates much less signal than comparable fluorescent...three luminescent proteins can be used together in cells. As Nano-lanterns are not subject to photobleaching...phototoxicity, they successfully imaged multiple subcellular compartments over several minutes using Nano-...concentrations of the compound can be added to the media. Cellular esterase then converts diacetyl coelenterazine-h...

...to visualize multiprotein complexes.  Fig. 2. Multiplex immunolabeling with subclass-switched recombinant...cryopreserved hybridomas, immortalized cells created by fusing an activated B cell that produces the antibody of...of interest with a myeloma cell. This RNA was then used to generate cDNA, which  was used as a template...of hybridoma generated mAbs is that if the frozen cell line becomes non-recoverable, the stock of the mAb...hybridoma stocks, without having to grow up the cells. This RNA could then be used to generate functional...immunofluorescence-immunocytochemistry (IF-ICC.) This method was chosen because cells transfected with a plasmid to express the target... could be screened in a high-throughput manner. Cells were labeled with both a hybridoma generated mAb...