...PMID: 25386923. PubMed Central PMCID: PMC4227841. 2. Young, Carissa L., Zachary T. Britton, and Anne S...doing some research, notably determining the subcellular localization and the number of disulfide bonds...

...light sensing proteins. Furthermore, during the first 2 weeks of life, larvae already exhibit distinct behaviors... light sensitive proteins (opsins) to manipulate cell activity, enables researchers to silence or incite...channels that allow charged particles to cross the cell membrane when exposed to light. Neuronal firing ...channel LiGluR both cause ion movement that results in cell depolarization. They therefore function as a neuron...in negative chloride ions which hyperpolarize the cell. It therefore functions as a neuron “off switch....They found that photostimulating the Kolmer-Agduhr cells was sufficient to trigger the symmetrical tail movement...forward swimming. Furthermore, only Kolmer-Agduhr cells on one side of the body required photostimulation...

...dissociation by bioluminescence resonance energy transfer 2, or BRET2. With binding of the drug, the alpha and... to environmental conditions, cells must be able to detect extracellular stimuli. One way they do so is...bound to the intracellular surface of the GPCR. Binding of a ligand to the extracellular surface of the...complex converts a signal received by a GPCR on the cell’s surface into action that can result in the initiation...number of signaling cascades, inducing different cellular responses. One of the biggest advantages of such...protein transducers. To use the TRUPATH system, cells are transfected with plasmids expressing the GPCR... and the appropriate Gγ-GFP2 and Gβ constructs. Cells are then treated with the luminescent enzyme substrate...

...development and therapy. Nat Rev Drug Discov (2017) 16(2):89-100. DOI: 10.1038/nrd.2016.238 Additional resources...identification of potential targets by CRISPR can accelerate the drug pipeline. High-throughput CRISPR screens...knock out, etc. – CRISPR can be used to generate cell line or animal models which have the precise disease...Since CRISPR has been adapted for use in primary cells, many tissue types, and across species, it enables... phenotype. CRISPR can also be used to generate cell line knock-ins of patient mutations which are variants...CRISPR has revolutionized the generation of knockout cell lines which has made this control step extremely...in a cancerous background and treat the parental cell line and the X knockout with your drug(s). If a ...

...remained active in up to 30% plasma. Figure 2: Human plasma longevity of SpCas9 vs GeoCas9. SpCas9...mammalian cells. Harrington et al tested GeoCas9’s editing skills by transfecting HEK293 cells with GeoCas9...choice for a number of model organisms: mammalian cells, yeast, drosophila, plants, worms, zebrafish, frogs... 45 °C 50 - 70 °C Genome Editing in Mammalian Cells? Yes Yes Longevity in Serum?* No Yes Table ...experimental results. GeoCas9 RNP editing in mammalian cells Besides being able to take some heat, GeoCas9 can...

...articles, such as: Sander JD & Joung JK, Nature Biotech, 2 March 2014. Or browse the articles related to the ...allows you to sort for GFP-positive cells and to enrich for those cells that were positively transfected....introduction of a mutation into the genome, how can cells with that mutation be selected/screened? A4: Before...can use a selection marker to select transfected cells (for example, plasmid with a puromycin resistance...paper). If you detect positive HR, isolate single-cell colonies, grow them up, then perform individual ...

... gamma (IFNγ) - and eliminate cancer cells. 2. Equilibrium - In a process analogous to the development...PMID: 25818339. 2. Curiel, Tyler J., et al. "Specific recruitment of regulatory T cells in ovarian carcinoma...activity, and consists of other cell types, especially immune cells and cells of epithelial origin. Suppressing...turns off T cell function upon interaction with APCs. Furthermore, tumor cells alter their cell-surface proteome...molecules needed for T cell effector function. Cancer cells also produce programmed cell death ligand (PD-L1... activity of CTLs and NK cells. These cells would normally kill cancer cells but are directed not to through...to use whole cell therapeutics consisting of various types of immune cells. Dendritic cells (DCs) are particularly...

...Almo, Institute for Protein Innovation  1:18:45 - 2:01:18 (end) - Panel Alex Tucker (Ginkgo Bioworks)...their curation of citable, linkable figures. Cause #2: Hidden “negative” data We all want to be able to ... characterised antibodies against all human extracellular proteins with community-based validation. They...

...teaching and mentoring others (see more in Figure 2). Starting early demonstrates not only your interest...application process so you can learn more! Figure 2: The many ways to prepare for a science policy career...example proposals and consider applying for their accelerator for some hands-on guidance.   Check out my webinar...

...establishing the dynamic range of your FRET assay (Figure 2). These labeling methods are freely interchangeable...PMID: 20161833. PubMed Central PMCID: PMC2802762. 2. Dumas, Anaëlle, et al. "Designing logical codon reassignment–Expanding...Mammalian Cells pCMV-DnpK dinitrophenyl hapten Mammalian Cells pANAP AnapRS Mammalian Cells Note:... Qi, and Marcel P. Bruchez. "Advances in chemical labeling of proteins in living cells." Cell and tissue...endogenous biomolecules in cells nonspecifically, and they cannot cross cell membranes, so they are best... ion channels, or cell surface receptors) or with experiments on permeabilized cells. Biarsenical labeling...these compounds cross cell membranes, thus enabling FRET measurements in intact cells. The Gradia Lab has...

...majority of deposited materials to study SARS-CoV-2 would be fully open and available to industry requestors...they grasped the crucial role Addgene plays in accelerating research. Yet this was still not enough to ensure...type of biomedical science research. Our work accelerates and enhances productive collaborations as well...distribution of plasmids for COVID-19 research is accelerating essential research in laboratories all around...scientists worldwide with Addgene’s traditional excellence through whatever lies ahead this year. We can...

... ligands can elicit varying degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic...fluorescent labels. In Nature Methods (Vol. 19, Issue 2, pp. 149–158). Nature Research. https://doi.org/10.1038... is technically cell-permeable, its use in live cells can sometimes result in cellular toxicity due to... Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental context...Additionally, cell permeability is a critical factor, as it influences the dye's ability to enter cells without...enabled the visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive...fluorophores to enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent...

...developed fluorescent-protein based DA sensors (Figure 2). The dLight1 dopamine sensor In 2018, Lin Tian’s ...the brain has been in vivo microdialysis of extracellular fluid (Darvesh et al., 2011). This technique... across the semipermeable membrane from the extracellular fluid into the perfused liquid. The probe then...detection of dopamine in flies, fish, and mice." Cell 174.2 (2018): 481-496.PubMed PMID: 30007419. PubMed...

...method relies on the fluorescence ratio between the 2 fluorophores, rather than on the fluorescence intensity...Fluorescence Activated Cell Sorting (FACS), allowing for enrichment of successfully targeted cells that can be used...investigating genetic mechanisms related to morphogenesis, cell fate specification, and human disease.     Sun,...

...make it easier to study these mutants.   Fig. 2: (A) Betacyanin is produced from tyrosine by the activity...graduate student studying plant development and cell-cell communication in the Nimchuk Lab in the Biology...untransformed seedlings or callus (undifferentiated plant cell culture), which is great for a wide variety of applications...

...Pringle’s former lab at UNC Chapel Hill. Bähler et al.(2) describe a modular collection of plasmids for a wide...insertion. The linear PCR product is introduced into the cell by direct transformation. A given insert will typically...

...E. coli: acoustic gene 1 (arg1) and acoustic gene 2 (arg2). The key difference between arg1 and arg2 vesicles...limit of detection for arg1 expressing ECN is 109 cells/ml, which is within the range of  the ~1011 commensal...

... last ~2-7 days at moderate levels. With regard to target cells or organism, some primary cells and differentiated...packaging plasmids into a viral production cell line, usually HEK-293 cells. When all of these viral plasmids ...secreted into the cellular media, making viral collection fairly easy. Producer cell media can be collected... rogue HEK-293 cell). This virus-containing media can then be placed on whatever cells you would like ...just zapping your cells? That really depends on two things. First, what type of cell or organism context...process of transferring foreign nucleic acid into a cell by a viral agent. Transductions are similar to transfections...processes which introduce foreign nucleic acid into cells by any means, but are unique in that they require...

...access this tool, set up an IDT account. Figure 2: Addgene's Analyze Sequence tool.   DNA sequence... repository of CRISPR screens Cellosaurus: A knowledge resource for cell lines IBEX Knowledge Base: A ...

... temporal activation of a transgene. Figure 2: FLEX Cre-Lox system.  WPRE The Woodchuck Hepatitis...about synthetic promoter AAVs for cell-type expression in retinal cells Resources on Addgene.org Find ...

... stabilization of unknown bacteria. Figure 2: Christina Mork collecting samples at the hot spring... life that remained unexplored. Using new single-cell sequencing approaches, the lab was able to eliminate...sample was separated into smaller samples of 5-10 cells each for sequencing, allowing for lower complexity...

...PMID: 30083608. PubMed Central PMCID: PMC6070312. 2. Stark, Jessica C., et al. "BioBits™ Bright: A fluorescent...on freeze-dried, cell-free (FD-CF) components. Making proteins outside of a cell - cell free extracts Typically... into a host cell (through a process called transformation), essentially giving the cell all the instructions...to dispose of the live cells. The alternative is to express the protein in a cell-free manner. This is ...biology, especially those at the molecular and cellular level. The reason for this is cost and complexity...electrophoresis to separate DNA or transforming cells with green fluorescent protein, these activities...transcription and translation. Unfortunately, having live cells in a classroom presents equipment needs, such as...

...structure-based framework engineering. Methods, 34(2), 184–199. PubMed PMID: 15312672. Inbar, D., Hochman...Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable...systems, and often do not fold properly once inside cells.  Developing the HA frankenbody To combat the issues... detects the widely used HA epitope in diverse cellular environments. They called this probe the HA frankenbody...previously proven to fold and function in living cells. To make these scaffolds specific to different targets...the tagged protein upon probe binding in living cells.  In their proof of concept experiment the Stasevich...mCherry, 4xHA tagged histone protein (H2B) in U2OS cells. They then showed that the HA frankenbody probe ...

...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...

...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 ...

...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...

...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, ...