...reagents and keep the cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and...however, you’ve collected the producer cells, you’ll need to lyse the cells to let the virions out. We recommend...particles that can transduce cells, which can vary based on target cells and can be altered by freeze/...several facilities nationwide where you can obtain excellent quality, high titer rAAV (including Addgene!),...come into contact with the rAAV and apply common cell culture practices diligently throughout the production...capsid genes.  In this helper-free system, HEK293T cells, expressing Ad E1A and E1B, can be transfected with...and then collect the supernatant and/or producer cells for further processing. What component(s) you need...

...at bioRxiv. AAV2 interacts with the packaging cell's cell membrane When packaging adeno-associated viral...of the packaging cells (Vandenberghe, 2010). Rather than being released into the cell culture supernatant... particles associate with the cell membrane and are taken into the cell via endocytosis. In our study,...AAV2 viral particles by our packaging cells by treating the cells with the endocytosis inhibitor sucrose...target cells is blocked following sucrose treatment. Moreover, several reports have shown that cells treated... yields approximately 1.8-fold without affecting cell viability. This effect appears to be serotype specific...difference between untreated and sucrose treated cells. Although we were unable to deduce the mechanism...

...manipulations on cells, and has potential use for a wide range of applications in cellular biology. Van ... several cell types is the lack of a rapid and quantitative editing assay. To help accelerate the expansion... of a microtubule-associated protein to regulate cell growth dynamics Article contributed by Klaus Wanisch... by Torsten Wittmann’s lab at UCSF to modulate cellular dynamics with light by modifying EB1. EB1 normally...of the modified EB1 protein are overexpressed in cells, microtubule growth can be switched off with blue...paper demonstrated that the migration of a cancer cell line equipped with photodissociable EB1 could be... opens novel ways in manipulating and studying cellular dynamics. The photoinducible control of protein...

...holes in the cell membranes of your sample to allow antibodies better access to intracellular targets. If...allowing antibodies into the cell where they could bind to intracellular epitopes. C) Permeabilizing with...method for visualizing molecules of interest within a cell or tissue. In that introduction, we broke down the...structure of your samples, the relationships between cellular components, and the accessibility of your target...cross-linkers work by (as the name suggests) cross-linking cellular proteins. Cross-linking is a chemical reaction...great for preserving your samples and maintaining cellular and tissue morphology. Formaldehyde is the most...ethanol, work by dehydrating and precipitating cellular components. This method of fixation is good for...

... for stem cells, or metastasized tumor cells, or alzheimers-affected neurons, or [your cell line of interest...critical to stem cell survival. Knowing which genes influence the survival of our cell culture is the foundation...minimal side-effects on healthy cells and maximal effect on unhealthy cells. Until recently, the human genome...ENCODE project, find enhancers in embryonic stem cells, and conclude with a targeted CRISPR screen that...essential transcription factor in embryonic stem cells (ESCs). Your ChIP-seq finds just over 13,000 significant...We start by infecting millions of embryonic stem cells (ESCs) with a lentiviral vector, an attenuated retrovirus...The result is a heterogenous population of stem cells, usually housed in a single flask. Through a bit...

...the degradation of over 600 cellular components ultimately leading to cell fragmentation and death (Elmore...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 ...sfCherry however did not initially fluoresce in HEK293 cells. To rectify this the Shu lab first improved the ...

...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...

...By taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...adenosine (A) to inosine (I, treated as G by the cell). Surprisingly, the team noticed that this editor...an inhibitor of base excision repair (BER), the cellular process that catches mutated U’s in DNA and ideally...cytosine editing between bacteria and mammalian cells Researchers in the Xueli Zhang lab were making similar...2021). Their GBE produced C → G edits in HEK293T cells with over 50% efficiency in 23 out of 30 target ...follow the same initial mechanism as in mammalian cells — by excising the freshly edited U bases — but E...the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com. Beyond...

... a big issue for cells, but it may confound research results or even kill your cells if your gene of interest...necessary to replicate plasmids and make sure the cells maintains it. Here, we'll cover the promoter, which... Because transcription machinery differs between cell types or organisms, promoters must be similarly ...variable. Bacterial promoters only work in prokaryotic cells and typically only in the same or closely related... were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc) require unique...complex, having fewer parts than those in eukaryotic cells. Some promoters are constitutively active and on... at will with a chemical, heat, or light. In the cell, promoters themselves are controlled by still other...

...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...antibody-encoding genes over time as the hybridoma cells divide. These changes can alter the antibody that...

...transducing the cell type under study. Many AAVs will show significant expression in a variety of cell types, ...optogenetics as tools to investigate the roles of certain cell types in locomotion. We have relied heavily upon...deliver chemogenetic/optogenetic constructs into our cells of interest. Although they have a small packaging...of serotype (which determines which tissues and cells an AAV can infect) can influence an AAV’s value ... tissue? Location (2): Where in the tissue/which cell type? Location (3): Traveling from the original ...AAV5, AAV6, AAV9 Pancreas AAV8 Photoreceptor Cells AAV2, AAV5, AAV8 RPE (Retinal Pigment Epithelium...-vectors/aav/aav-guide. Where in the tissue/what cell type? In the CNS, for instance, there are marked...

...editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635-5652.e29. https...efficient prime editors. Cell, 186(18), 3983-4002.e26. https://doi.org/10.1016/j.cell.2023.07.039 Lin, Q.,...correct only a minority of those variants in most cell types. But prime editing, a CRISPR technique developed...unedited DNA. The heteroduplex is resolved by the cell’s mismatch repair system, and more recent prime editing...editing demonstrated ~20-50% efficiency in HEK293T cells with 1-10% indels. Since then, further innovations...type of edit, genomic context, delivery strategy, cell type, and goal of the experiment can all impose ...nicking the non-edited strand, they can cause the cell to remake that strand using the edited strand as...

...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....

...proteins in ten ddifferent cell types, including difficult-to-manipulate primary cells, achieving knockdown ...stable cell lines overexpressing TRIM21, finding that TRIM21 overexpression did not affect cell proliferation...electroporated cells re-adhered to the culture plate within 4 hr of treatment. In some primary cell lines, endogenous...proliferation. These cells also did not contain changes in expression of TRIM21-associated genes, indicating...multiple methods for TRIM-Away delivery. For small cell populations, co-microinjection of TRIM21 protein...protein and the target antibody works well. In larger cell populations, you can deliver these components together...together using electroporation. In these bulk cell experiments, Clift et al. observed no stress or damage...

...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...

...mammalian cells. dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with ...Near-Infrared Fluorescent Proteins in Mammalian Cells. Cell Chem Biol 24, 758-766 e753 (2017). Baloban, M...developers and users, BV is present in eukaryotic cells as an intermediate of heme metabolism (Shemetov ...to GFP-like probes by supplying a single gene to cells. Still in bacteria, which do not produce BV, an ... to consider effective brightness of NIR FPs in cells, in addition to their molecular brightness (a product...the concentration of BV-containing molecules in a cell and depends on the efficiency of BV incorporation...biosensors can be imaged at spatial scales from sub-cellular to whole-body in small animals, such as mice (...

...modified zygotes or embryonic stem cells. Modified embryonic stem cells are injected into a blastocyst which...engineered cells. Similar to transgenic mice, knockout mice are engineered during the embryonic stem cell stage...not all cells in the blastocyst were successfully edited by the injection of embryonic stem cells. Also,...controls the cell types in which the conditional recombination can occur based on the cells in which Cre... interest into a zygote, or into embryonic stem cells that are then injected into a blastocyst. There ...of interest into a zygote or into embryonic stem cells. Non-homologous recombination incorporates this ...targeting vector is electroporated into embryonic stem cells and this will insert the gene at a specific site...

...illumination of neighboring cells evokes action potentials (APs) in patched cells expressing wild type CoChR...techniques for live-cell imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence...distribution of clusters without accumulation in subcellular compartments such as nuclear speckles. Furthermore...OptoSTIM1), caused two-fold faster changes in intracellular Ca2+ levels than the original version containing...CRY2clust should prove a useful tool to further probe cellular function. The CRY2clust Plasmid can be found here...28646204. soCoChR: A novel opsin that enables single-cell optogenetics Article contributed by Meghan Rego ...wild type CoChR, soCoChR localizes primarily to the cell body with very little signal detected along the ...

...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 ...

...offer scientists a simple yet powerful way to tag cellular proteins and investigate protein localization,...continuously synthesized and degraded within the cell, so at any given time, an FP-chimeric protein may...Since emission of green light is more phototoxic to cells, most scientists working on in vivo imaging models... this is not ideal for experiments in mammalian cells. Dendra2 is another monomeric PA-FP which unlike...tetrameric. Tetrameric PA-FPS are better for whole cell imaging and studying organelle trafficking. Monomeric... chimeric protein will oligomerize, disrupting cellular function and localization, or leading to protein...the photoactivation step can actually harm your cells before your experiment even begins! If you are performing...