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...epitopes likely to be presented by T cells to elicit an immune response. T cell epitopes are found in either ...that take place within an antigen-presenting cell like a T cell. It is also not yet possible for these algorithms...traditional 2D assays with immune cell lines or peripheral blood mononuclear cells (PBMC) in 3D models of the...CD4+ or CD8+ T-cells or monocytes. PBMCs can be further differentiated into dentritic cells or macrophages...impurities in the formulation. Efforts to predict T cell epitopes focus on characterizing those epitopes ..., that are commonly used for the prediction of T cell epitopes based on the amino acid sequence of the...lymphatic system. PBMCs consist of various immune cells, and can be used as a whole or can be used to isolate...

...the surface of basophils and mast cells, two types of white blood cells involved in allergic reactions. ... is important because it determines what immune cells and molecules are recruited by the antibody to help...each of these functions: Recruitment of immune cells that express receptors that recognize the Fc portion...special receptors that transport antibodies through cells and into different body compartments, such as into... during a primary immune response because all B cells begin by expressing IgM. IgM has a pentamer structure...coexpressed with IgM on the surface of most immature B cells, its function is unknown. It has a monomer structure...Isotype switching Isotype switching occurs when a B cell changes which heavy chain constant domain it pairs...

...for neurons. Non-neuronal cells do this too. It's just that for non-neural cells, ion-based communication...between many cells (through gap junction proteins which connect the cytoplasm of two cells) can also affect...more depolarized (positive) the cell is, the more embryonic, stem cell-like, or cancerous it is (Levin...happens at a slightly slower scale.  All living cells with a membrane possess an electrical state. This...charge between the inside and the outside of the cell. This imbalance is maintained by ion channels and...proteins cx43 GJB1 GJB2 Bioelectric states can alter cell types and patterning There is mounting evidence ...more polarized (negative) the action potential of a cell, the more differentiated and quiescent it is, and...

... quantified for individual cells, and the number of 'normal' looking cells can be counted. This assay ...living cells. Strikingly, the OSER assay reveals that tagRFP does not act as a true monomer in cells (Constantini...tolerated in some applications (e.g. labeling of cells or tracking promotor activity), but it is problematic...fluorescent proteins interact or oligomerize in cells? This question is largely unanswered. Ideally, fluorescent...act as inert modules that fail to homodimerize in cells. To understand how well fluorescent proteins approach...proteins could properly form microtubule networks in cells. Other efforts tested whether FP fusions to connexin...present in EGFP. Constantini et al, (2012) developed a cell-based assay that does a better job of detecting ...

...endocrine cells. It follows that the specific targeting of these voltage indicators needs to be cell type ...electrical events! Voltage indicators are voltmeters for cells! In this blog we will review how these indicators...related to the electrical impulse of the cardiac cells. To study this phenomenon you will likely want to...the voltage change across the membrane of cardiac cells. Similarly, neuronal signaling is carried out by... electrical charges propagated through multiple cells to do things like translate perceived signals to...Understanding neuronal pathways and the function of certain cells depends on the ability to monitor their signals ...The research tools for studying voltage within a cell are genetically encoded voltage indicators (GEVIs...

...Expression of firefly luciferase from a cell-free mammalian cell lysate Marcel Bruchez 108542 pLenti-EF1a-Luciferase-IRES-Blast-WPRE...kDa, respectively). And they are not secreted, so cells must be lysed or the substrate must be delivered...expression of luciferase in the cytoplasm of mammalian cells Peter Cockerill 212936 pGL3 Basic Vector Firefly... viral infection efficiency, to study changes in cell physiology, or as a reference level of expression...Expression of Gaussia luciferase; GFP is expressed if cells are infected with virus Christopher Buck 98580 pLX304...

...to enable three-feature cell targeting. C) Example of viral targeting of cells expressing Cre AND Flp,...parameters. Figure 1: Examples of intersectional cell population definitions using recombinases. A) Cre...adeno-associated virus (AAV)-borne payloads to be expressed in cells based on a doubly-specified combination of genetic...Neve R, Huang ZJ, Deisseroth K. 2014. Targeting cells with single vectors using multiple-feature Boolean...Cortex Reveals Local and Long-Range Circuit Motifs. Cell Rep. 28(13):3450-3461. PubMed (Link opens in a new...

...Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific expression of molecular tools...in their cell bodies. Optical stimulation in a different brain area can then reveal the cells bodies of...with AAV: 1. Getting your viral vector into your cell of interest: AAV tropism and route of administration...transduction, the viral capsid proteins bind to the target cell’s surface proteins and this interaction determines...efficiency of the AAV serotype used for a specific cell type. Find some transduction characteristics of ...by restricting expression to genetically defined cell types. Specific transgene expression can be also...different approaches can increase the specificity of cell targeting even further. For example, injecting AAVs...

... of cell types, so they’re ideal for work with primary cells or other difficult-to-transfect cells. Lentiviral...dividing cells. Another type of vector, based on adenovirus, could infect non-dividing cells, but without...ability to transduce both dividing and non-dividing cells. Lentiviral vectors are the vector of choice for...lentivirus HIV-1, well-known to infect non-dividing cells. The first lentiviral vector system consisted of...viral envelope to dictate the tropism, or types of cells the vector could infect. Finally, the transfer plasmid.... After co-transfection of these plasmids, 293T cells released transgene-containing lentiviral particles...adeno-associated viral vectors can also target non-dividing cells, lentiviral vectors can carry much more genetic ...

...this approach is limited to protoplast cells (plant cells whose cell wall has been removed). Another common...expression is dependent upon the cell type in question. In plant cells, gRNA expression is achieved by ...are necessary to enable genome editing in plant cells.   This blog post will present a general overview...are necessary to use the CRISPR system in plant cells. Like other model systems, expression of S. pyogenes...gRNA) is sufficient to modify the genome of plant cells. The structure of the gRNA (composed of a ~20 nucleotide...increase translation in a particular plant species or cell type (Belhaj et al., 2013). Nuclease dead Cas9 (...used to activate or repress target genes in plant cells, respectively. Cas9 expression is commonly driven...

...introduced the reporter into the cells, you’re ready to edit. These cells are then transfected with several... up, all successfully transfected cells will express RFP while cells containing the reporter gene that...using the RFP:GFP ratio, edited cells can be sorted from unedited cells. In an experiment that targeted...CRISPR experiment was successful in your organism or cell type? You can use DNA sequencing or other molecular...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct...simply changing the gRNA used when transfecting your cells.  A plasmid that contains Cas9. The Madison lab ...NHEJ as well. The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  ...

...Time to expression AAVs have to infect cells, hijack host cell machinery, and synthesize a second strand...that can be used to express genes of interest in cell and animal models. AAVs are non-pathogenic, do not...be specifically targeted to a number of different cell types even when systemically administered in an ...ranging from transient gene expression in a particular cell type to CRISPR genome editing, optogenetics, and...application of AAV is delivery of genetic cargo to a host cell. In order to make room for a gene of interest, AAVs...allow the virus to infect (or not infect) different cell types with some specificity. There are a lot of ...ability of AAV to drive gene expression in specific cells and/or tissues is one of its biggest advantages....

...can actually infect cells. These assays typically involve infecting a target cell line with your virus...distinguish between a cell that has had one viral particle integrated into its genome and a cell that has had ...arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested...move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure to...viral copies that have integrated into the target cell’s genome. Physical titer The two most common assays...functional titer uses FACS to count the number of target cells that are positive for vector encoded transgene expression...resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions of a viral ...

...targeted One Many Sample type(s) Cell lines Cell lines, primary cells Table 1: Key differences between... of cells (~5x107). Together, these two requirements prevent the use of CAPTURE with primary cells or ...need to create stable cell lines and allows CAPTURE 2.0 to be used with primary cells. Find plasmids for...CAPTURE method requires the creation of a stable cell line that co-expresses three components: dCas9 ...drawback of the original CAPTURE method is that a new cell line has to be created for each genomic loci of ...across multiple targets because different CAPTURE cell lines will have varying levels of dCas9 and gRNA...or rare cell populations. Key components of the CAPTURE 2.0 system To improve upon the original CAPTURE...

...PV-positive cells out of all BiPVe3-dTom cells. C) Confocal stack of a biocytin-filled cell expressing ...particles in HEK293T cells with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260). HeLa cells were infected...Scheme for two-photon (2P) imaging and loose-seal, cell-attached recordings in primary visual cortex (V1...contains everything needed to replicate inside a host cell but with space to insert your genetic cargo of choice...promoter drives robust gene expression in mammalian cells. Flexible tagging: Choose untagged or add a versatile...stable: A puromycin resistance gene makes stable cell line creation a breeze. Modular by design: Key elements...step-by-step protocols for viral generation and stable cell line creation (Figure 2). Looking for more? Explore...

...specific cell type rely on transgenic models that express recombinases like Cre or Flp in their cells of interest...entry into the cell (Dudek, AM. 2018).  If the serotype you are using transduces cells that you are not... an ATG start codon to prevent transcription in cells where the recombinase was not present (Kuhlman, ...recombination that can lead to leaky expression in cells that do not express Cre or Flp. Here at Addgene ...recombined/functional transgene that can be expressed in cells where Cre is absent. Additionally, we have experienced.../Flex vectors and leaky expression can occur in cells that don’t express Cre or Flp, what can we do about...volume needed to achieve adequate expression in your cells of interest.  The AAV serotype used can exhibit ...

...yielded editing of 80–90% in human cell lines and primary cells. Although cell entry of a Cas9 ribonucleoprotein...post: Peptide-assisted genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic...antibodies Peptide-assisted genome editing for primary cells By Susanna Stroik Even with the advances in CRISPR...CRISPR technology over the past decade, primary cells remain difficult to genetically edit due to toxicity...Genome Editing (PAGE). They fused the Cas enzyme to cell-penetrating peptides and co-incubate it with an ...including in clinically-relevant lines such as CAR-T cells. Plus, this method is fast! These editing frequencies...even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and SV40...

...of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can...dividing and non-diving cells while retroviruses can only infect dividing cells. When choosing the appropriate...to ensure that the proper cell line is used; several common laboratory cell lines including 293 were immortalized...can infect dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids...transfecting four different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and...appropriate gene delivery system, the specific target cell or tissue needs to be considered. Q2: Can I use...investing time and resources into making a stable cell line. When expressing a viral construct transiently...

...issues with tissue autofluorescence, cell heterogeneity, and/or cell position changes (To et al., 2016, ...apoptosis in HeLa cells, zebrafish embryos, and Drosophila. They treated HeLa cells expressing FlipGFP...cleavage by executioner caspases. Failure for cells to die or cells dying at the wrong time and/or place can...Apoptosis or “programmed cell death" plays a pivotal role in an array of biological processes including...including development, the immune system, and cell turnover. Apoptosis is a highly controlled process that ...over 600 cellular components ultimately leading to cell fragmentation and death (Elmore, 2007). To study...sfCherry however did not initially fluoresce in HEK293 cells. To rectify this the Shu lab first improved the ...

...within an E. coli cell, but vectors can be widely utilized across many different cell types and each one... the yeast cell. These elements control not only the number of plasmids found in each cell, but also whether...are eukaryotes and thus contain complex internal cell structures similar to those of plants and animals...components to allow shuttling between E. coli and yeast cells. To be useful in the lab, the vectors must contain...replication (ORI) and a means of selection in yeast cells, in addition to the bacterial ORI and antibiotic...determines plasmid copy number within the bacterial cell. Similarly, the specific ORI elements included within...plasmid) allows for 50+ copies to stably propagate per cell. The copy number of these vectors can also be controlled...