...on or in these cells: antibodies can bind to proteins on the cell surface, or the cell membrane can be...same cell simultaneously as well as compare between samples. Learn more about flow cytometry. Cell and ...antibodies are produced by B cells. They bind to proteins on the surface of extracellular pathogens such as parasites...fusing B cells that have been extracted from animals injected with protein antigen to myeloma cells. The ... measurement of protein expression in cell lysates, whole cells, or tissue samples. Many experiments that...shows that a similar concentration of cell lysate was added to each well. Image from Sun et al., 2010. ...heterogeneous mixture - for example, cell lysate or media from cultured cells. ELISA can be used to quantitatively...

...quiescent cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting...therefore titers obtained in one cell type may not translate well to other cell types. To overcome this issue...Love your cells and they'll love you back A successful lentiviral infection begins with your cells, both ...both your packaging cells and your target cells. Poor tissue culture practices can lead to low titers and... promote cell growth. Overgrown cultures stimulate quiescence, the resting phase of the cell cycle; quiescent...reaching 108 cells per ml before the media becomes cloudy. Mycoplasma compete with host cells for nutrients... your producer cells with fresh medium. Daily medium exchanges help alleviate cellular stresses associated...

...producing cells. After FACS, return the cells to doxycycline-free media and expand the cells. Use a subset...CMV-gvpNJKFGWV plasmid into 70%-confluent well of HEK cells in a 6-well dish and change the media every day ...white inclusions in cells with GVs via phase contrast microscopy, GVs visible in cells via TEM, and purified... heterogeneity required single cell screening to isolate a clonal cell line capable of producing ultrasound...able to express Anabaena GVs in mammalian cells without the shell stiffening protein GvpC, which allowed ...expression homogeneity, as the cells could be batch-sorted to isolate a polyclonal cell line with strong gas vesicle...transient transfection in rtTA+ cell lines (such as HEK293-TETOn cells), or they can also be integrated...

... your cell type of interest, while miRNA target sites can be used to prevent expression in cell types ... promoter/enhancer strengths in the cell type of interest as well as the type of transgene delivered: ...endothelial cells in rodent CNS AAV-PHP.V1 shows higher transduction in CNS endothelial cells (60%) and ...endothelial cells in the retina and spinal cord (Krolak et al., 2022). Systemic capsids for broad cell type ...delivery across species. Cell, 184(19), 4919-4938.e22. https://doi.org/10.1016/j.cell.2021.08.028 Vormstein-Schneider...system (PNS). (Capsids, if you recall, are the outer shell of an AAV vector, which determine the vector’s tropism... discuss how systemic capsids are engineered, as well as highlight some considerations for their use. ...

... cloned into E. coli cells, isolated colonies will be lifted onto nitrocellulose membranes and exposed... proteins directly onto the nitrocellulose for subsequent probing for cell-binding activity.  First, cut...with live host cells Now you can proceed with incubating this membrane with your cells of interest and...to bind their target cells, pulling them out of solution. Wash your membranes well with PBST to remove ...remove unbound cells. To detect bound cells, simply lay each membrane on an agar plate selective for your target...appropriate cell growth conditions. Note: if using fast-growing bacteria like E. coli or Salmonella, or any...any cells that bind strongly to nitrocellulose, over-incubation of plates can lead to high background that...

...embryonic kidney (HEK) cells originated from? Or why they are such a commonly used cell culture model? In ...isolating the cell line all the way to modern applications. What the HEK is it?   HEK cells were first ...inconsistent with kidney cells (Shaw et al.). Since then, transcriptome studies of the cell line have determined...for?   As one of the older cell lines in the research community, many cellular research tools have been ...any unwanted material (HEK cells, etc.) and are ready to use on another cell or organism. This pipeline...where HEK cells may be particularly useful due to their intrinsic properties. HEK-293 cells are tumorigenic...researchers using the cell line. What the HEK is wrong with it?   All cultured human cell lines are susceptible...

... subset of T-cells, but it is unclear if enough T-cells could be reached to ablate a well-established ...infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read on...1 reservoirs HIV-1 infects cells in the immune system, notably CD4+ T-cells, and eventually leads to acquired...promoter-luciferase constructs. In multiple HIV-1 latent T cell lines, as well as a latent microglial line, treatment with...percentage of cells with activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that... genome from every infected cell and 2. prevent reinfection of those cells. Kaminski et al. designed gRNAs...Kaminski et al. observed no negative effects on cell viability, cell cycle progression, or apoptosis with Cas9...

...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...differentiation.   Fig. 2: Single cell RNA-seq clustering of cells transduced with the TF pooled library... 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...

...determine cell cycle length by examining the ratio of blue to red cells. Cells undergoing rapid cell cycles...engineered C. elegans cell lines to express wrmScarlet1-10 unattached to any other cellular protein. They used...’s difficult to observe the cell cycle in vivo, particularly if the cells are migratory or in deep tissues... BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations of IL-3. Cells grown...proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in media without IL-3 show...H2B-FT consistently predicted the cell cycle speed across these different cell types. In one example, H2B-FT...expressed in murine B cells (BaF3), which depend on IL-3 to proliferate. The cells cultured with a low ...

... target to specific subcellular regions, laser ablation can damage other cellular structures, and genetic...demonstrated its use to probe cell mechanics, vesicle transport, and cell division, but given the long ...GTPase, leading to behaviors like lamellipodia or filopodia formation, cell contraction, or other morphological... expressed in a monolayer of endothelial cells, showing cell protrusions and overlaps (arrows) after blue...2016). Rho GTPase signalling in cell migration. Current Opinion in Cell Biology, 36, 103–112. https://doi.org...Optogenetics gives you the power to control cells or organisms with the flip of a switch. You may be ...optogenetic tools to control the cytoskeleton and cell membrane dynamics. First, a quick refresher.  The...

...infecting most cells, but not all. For example, LDLR expression in hematopoietic stem cells, T cells, and B cells... host cell. Some of these envelope-receptor pairings are broad, allowing delivery into many cell types...delivery to only a few cell types.  If you’re looking for more control in the cell types your lentiviral...protein causes cells that it infects to fuse together and form syncytias that cause the cells to die. Cytotoxicity...Cytotoxicity can also be cell type dependent: if a cell has few receptors for a specific envelope protein...lipoprotein receptors (LDLRs) on a cell surface to endocytose into the cell. The reason VSV-G is so commonly... cells is low. Infection of these cells with VSV-G pseudotyped virus requires the upregulation of LDLR...

...Fluorescent proteins in cellular organelles: serious pitfalls and some solutions." DNA and cell biology 32.11 (...localize to chemically distinct subcellular environments, including the organelles that compose the secretory...numerous cell biology assays. However, there has been a pervasive view that most FPs in a cell will fold...fluorescent proteins for subcellular environment. Currently, she is a SPIRE postdoctoral fellow at the University...fluorescent fusion proteins in cell biology."Current Protocols in Cell Biology (2005): 21-4. Pubmed PMID...Lee. "Fluorescent proteins: a cell biologist's user guide." Trends in cell biology 19.11 (2009): 649-655...-fusions on quantitative imaging experiments and cell physiology in general. Pitfalls of Fluorescent Protein...

...one side of the cell and released again at another side of the cell after intracellular transport without...typically used for the trafficking of organelles, such as mitochondria, as well as macromolecules like actin and...diffusing in the extracellular space; they also have low immunogenicity and cellular toxicity. AAV vectors...tracing has the added benefit of only labelling the subset of infected cells that also express Cre. Another ...requires both antero- and retrograde labelling, synaptic restriction as well as controlled monosynaptic and ...via individual neurons. Neurons are composed of a cell body, with dendrites receiving incoming information...axon sending information onwards to other neuronal cells. Synapses at the terminals of axons form connections...

... into cells in the lab. The introduction of foreign DNA or RNA into bacteria or eukaryotic cells is a ...application of an electrical charge to cells, to increase cell membrane permeability and thus transformation...introduce foreign DNA into eukaryotic cells, like mammalian cell lines. This can be done with lentiviral...the genome and stable cell lines, where foreign DNA is incorporated into the cell’s genome and is thus passed...through cell division. You can find all kinds of different lentiviral and AAV plasmids as well as ready-to-use... pokes holes in the cell membrane so DNA can be introduced directly into the cell. Microinjection requires...multiple ways foreign DNA can be introduced into cells including transformation, transduction, conjugation...

...model systems: either human cells or mice. In a recent issue of Cell Stem Cell, Musunuru, Chad Cowan and...done in stem cells, we can differentiate those mutant and wild type stem cells into any cell type relevant... the mutant cell lines that one needs to do disease modeling in stem cells. Addgene: Tell me about the...system for generating human stem cell-based disease models." Cell Stem Cell. 12, 238-251 (7 February 2013...homology-directed repair by flooding the cell with a mutant template and fooling the cell into using that template for...FokI endonuclease domain as well as fluorescent proteins used to sort the cells after the plasmids have been...unique aspects that make it especially well-suited for use in stem cells. (See the complete protocol at StemBook...

... the single cell level), are separated by, for example, cell sorting. The separated cells are subsequently...more than 8x107 successfully targeted cells. Depending on the cells used and the readout, this could be ...My group works on immune cells in different contexts. Most of the immune cells we study are migratory, ...CRISPR modified cells are transferred into an animal and the behavior of the transferred cells is followed...with an easily measureable cellular phenotype (e.g. survival of a cancer cell as a cytotoxic drug is added...genes essential for West-Nile-virus-induced cell death." Cell reports 12.4 (2015): 673-683. PubMed PMID:...With these screens, the investigator generates a cell population where all genes are knocked out at the...

...transgenic cell lines? One of the major uses of the piggyBac™ system is to create stable transgenic cell lines...efficiency is likely to be cell line-dependent, and should be tested in the cell line of interest. Importantly...If the cell’s chromatin structure undergoes significant rearrangement (such as when a stem cell initiates...manipulations in animal models or cells lines, including stem cells, without leaving behind any accessory...applications, including making transgenic cell lines and genetically-modified cell lines. Furthermore, the use of...molecular and cellular biology and brings 10 years of research experience with stem cells to Transposagen's... in Human Pluripotent Stem Cells: Choosing the Correct Path.,” J. stem cell Regen. Biol., vol. 1, no. ...

...to the well (either directly or by binding to an antibody that is coating the bottom of the well), and ...coating the wells to capture antigen from a sample, whereas antigen is bound directly to the wells in a direct... the wells are typically already coated with the antibody. Then, you add your sample to the wells, and...sample are bound to the wells of a plate instead of an antibody. Coating the wells with a purified protein...ELISA well As mentioned above, the measurement of the concentration of proteins in an ELISA well is based...the reaction, turns the solution yellow. The absorbance of each well, which is measured using a microplate... are used to detect proteins within a 96- or 384-well, flat-bottomed plate. The basic principle of an ...

...contaminating your transduction with the packaging cells. Packaging cell debris can be removed either by filtration...-500 g). After removing packaging cell debris, virus can be pelleted by ultracentrifugation (75,000 - ...ultracentrifugation, virus may appear as a white pellet. The pellet can then be rinsed and resuspended in cold... you can reduce the volume of cell culture medium on the packaging cells immediately following transfection...efficiency in cells that are sensitive to the toxicity of polybrene (e.g., hematopoietic or primary cells), and... the packaging cells after 1–3 days of selection. A lower percentage of surviving cells may be the result...adeno-associated virus transduction of cells." American Journal of Physiology-Cell Physiology 291.2 (2006): C386...

...restoring mCherry expression. Cells can then be sorted using Fluorescence-activated cell sorting (FACS) and the...easily determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry...expressed in cells. mCherry is constitutively expressed to identify successfully transfected cells. C-to-T ...eGFP and mCherry positive cells by the number of only mCherry positive cells (Martin et al., 2019). ...TREE) to purify single base edited cells without the need for single cell isolation and downstream sequencing...fluorescence and cell sorting using FACS which could limit their use across different cell types and systems...sgRNA targeting the GFPACG to the cells. As expected, only the cells that contained the cytidine base ...

...be different for each transduced cell, there’s no telling which cells may harbor these unwanted interruptions...purposes, like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing, and introducing... chromosome of the cell. This integrated viral genome is then part of the cellular DNA… forever!  This...viral entry into the host cell.  Transfecting these three plasmids into cells enables the production of...lost through cycles of cell division, though the rate of loss greatly depends on cell turnover. Why use ...expression in non-dividing cells IDLVs can also be useful for transducing non-dividing cells, as they avoid the... transduced cells and quantifying the integrated viral DNA along with a reference cellular gene. When ...

...Mammalian Cells pCMV-DnpK dinitrophenyl hapten Mammalian Cells pANAP AnapRS Mammalian Cells Note:...intervening proteins, lipid bilayers, organelles or other cellular impediments. FRET has been used in many...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...-72 Intact Cells Cy3NTAc Poly His  44 35-63 Plasma membrane protein, permeabilized cells  Cy5NTAc ...-70 Intact Cells Cy3NTAc Poly His  63 50-90 Plasma membrane protein, permeabilized cells  Cy5NTAc ...

...Non-Dividing Cells Transient Gene Expression Low Tropism for Mouse Cells and Hematopoietic Cells Low Packaging...cargo” to target cells and organisms, and when that cargo is designed to make you sick…well, you feel it....fuses with the target cell’s membrane, allowing the viral particle access into the cell. The viral envelope... of cell the virus will target and fuse with. In nature, this determines which organisms and cells they...viruses can be engineered to be cell type specific. Once inside the host cell, the virus unleashes its cargo...wide range of cell types that are typically ‘difficult to transfect’, such as primary cells. They can also...Viruses as biological tools In nature, viruses infect cells and organisms (which we refer to as hosts) via viral...

...Calcium phosphate Cells Transformed cell lines Transformed cell lines Transformed cell lines Cargo Nucleic...identify and enrich positive cells or a selection marker to generate stable cell lines. Transient or stable...and enrich positive cells. Likely contains a selection marker to generate stable cell lines. Packaging and...wide variety of mammalian cell lines. Useful for difficult to transfect cell types and can be used in ...protein are degraded within the cell.   Delivery methods for mammalian cell lines As mentioned above, the...components into your target cells. Delivery of these components into mammalian cell lines is quite broad and...Electroporation / Nucleofection Microinjection Cells Many types Single cells Cargo Nucleic acids or protein Nucleic...

...With regard to target cells or organism, some primary cells and differentiated cell types are picky when...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...viruses as well. Transduce on! Resources Resources on Addgene.org Generating Stable Cell Lines with ...process of transferring foreign nucleic acid into a cell by a viral agent. Transductions are similar to transfections...

...prevent cell death. One of the cool things about amp is that it's exclusively toxic to dividing cells. The... continuous cellular processes (e.g., protein synthesis), while amp only affects the cell wall synthesis...got amp? Well, amp is less stable than its counterparts and is also known to produce satellite colonies...decorative" title="Check icon" %} Satellite colonies Kanamycin Excellent No Bacteria and eukaryotes (G418...Check icon" %} Spectinomycin Excellent No Just bacteria Not SHuffle cells {% icon icon_set="fontawesome...icon" %} Zeocin Excellent No Bacteria, plants, and eukaryotes Not Tn5-containing cells {% icon icon_set..., penicillin, and is a beta-lactam that inhibits cell wall synthesis. The AmpR (amp resistance) gene produces...

...Winslow’s lab to label cells after cell-cell interactions. Using G-baToN, “sender” cells express GFP on the... and “receiver” cells express an anti-GFP nanobody on the cell surface. When the cells interact, GFP is...the receiver cell, and the receiver cell turns green. This system can be used in many cell types including...engineering with single-pot assembly Tracking cell-cell interactions New auxin inducible degron system....brgcm3sw     New system developed to track cell-cell interactions By Jennifer Tsang GFP-based Touching...including endothelial cell-pericyte, neuron-astrocyte, and cancer-stromal cell pairs.   Figure 2: Schematic...of the interaction between the sender cell and the receiver cell. Image from Tang et al., 2020.   ...

... of cell types in the nervous system. While certain cell types, such as principal excitatory cells and...neuronal cell types involved in neurodegenerative diseases, the Cole Lab selected promoters for cell types...great performance in cellular functional imaging and in protein fusions. The Gadella lab has deposited all...with edited “pools” of cells, eliminating the time-consuming step of single-cell isolation. Agudelo et ...gene. Koo M, et al. Cell Systems. 2016. PubMed PMID: 28189581 Peters J, et al. Cell. 2016. PubMed PMID:...generation Deriving iPSCs (induced pluripotent stem cells) through the expression of transcription factors...the established model for reprogramming somatic cells. A common method for generating iPSCs to express...

...linkage of intra- and extracellular environments. Found on nearly every cell in the human body, integrins are...array of processes, including cell adhesion, migration, and signaling, as well as immune response, tissue... that protrude from the cell membrane, physically connecting the extracellular environment and the cytoskeleton...ligand binding, mediating cell adhesion and migration, initiating intracellular signal cascades, and allowing... the extracellular matrix. During immune responses, this same conformation encourages immune cell adhesion...tensions before they would enable cells to spread, resulting in cells that elongated and morphed asymmetrically...on the BJ-5ta fibroblast cells, integrin αVβ1 was critical in initiating cell spreading and two other ...

...matters. Well, it turns out this isn’t the case for using AAV. When it comes to infecting cells, titer,...Making contact with a cell Although an individual AAV particle’s ability to infect a cell is dependent on its...construction and the cell it’s infecting, getting these particles to infect cells is also very titer-dependent... particle to infect a cell, it has to come into physical contact with that cell. Since there is a limited...of surface area on each cell, the way to get more AAV in contact with the cell is to increase the AAV ...between the cell and any individual AAV particle is rare. But simply getting into the cell may not always...particles that have entered the cell now have to travel up the axon, through the cell, and potentially spread ...