...that already do mammalian cell culture. HEK-293T cells are the most common cells used in viral production...versus insect cells for viral production Consideration Mammalian cells Insect cells Viruses AAV ...type of cells that will be used to make the virus. Viruses can be produced in many different cell types,...produced in bacteria cells. However, some viruses can be produced in multiple different cell types, including...you make an informed decision. Mammalian cells Mammalian cells are a great option for viral production,...production in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus...using qPCR or digital droplet PCR. Insect cells Insect cells are a less common option for the generation...

...types of primary mouse cells, including neuronal and gut stem cells, dendritic cells, embryonic fibroblasts...fibroblasts, glia cells and neurons, and cell death post-transduction is low. Neuronal stem cells transduced... Primary cells recapitulate the natural biology of a cell type of interest better than immortalized lines...introduce a gene of interest into primary cells, so most primary cell lines require viral infection. A new ... primary cells. What if, instead of supplying a DNA construct, they could simply induce cells to take ...primary cell types using Cre and various loxP reporters. In both mouse and human embryonic stem cells, they...cultured mammalian cells by osmotic lysis of pinocytic vesicles. Okada CY, Rechsteiner M. Cell. 1982 May;29(...

...spread from this cell to synaptically connected cells (meaning, it can spread to cells that are projecting...infect a single "starter" cell, but new virus cannot spread to any neighboring cells. To enable monosynaptic...provided to the starter cell in trans , via delivery by an AAV. Since this starter cell now has all the viral...projecting to the starter cell). Once in the secondarily infected cells that lack the G protein, the virus ...connected cells. To further refine the targeting of G-deleted rabies to specific starter cells, the tropism...G-protein is delivered to the starter cell. (2) The starter cell now expresses TVA and G-protein. EnvA-pseudotyped...is delivered to the starter cell, and specifically targets the starter cell via the EnvA receptor. RABV...

...and spread of cancer cells. Besides these stromal cell modifications, stromal cells are also constantly...how cancer cells influence microenvironments and vice versa, let’s take a look at cancer cell metabolism...inflammation, a noteworthy cell type in the tumor microenvironment is the immune cells. The contradiction is...injecting breast cancer cells into mice, exosomes expressed specific integrins (cell-adhesion proteins) targeting... be transferred to other cells, inducing epigenetic changes in targeted cells. In this case, exosomes ...Friendly” microenvironment cells Not all is bad news – we also have “friendly” cells in microenvironments! ...Take myoepithelial cells for instance. They retard tumor formation by inhibiting cell growth, invasion,...

...produced using complex mixtures derived from cells or using live cells. There are several types of expression...consists of transfecting cells with your DNA template of choice and allowing these cells to transcribe, translate...demanding cell culture conditions compared to other systems. Insect expression systems Insect cells can also...coli or yeast cells. The only problem with baculovirus systems is that the infected host cell is eventually...eventually lysed. Cell lysis halts protein production, but there are non-lytic insect cell expression systems...Sf21, Hi-5 cells)  that allow for continuous expression of genes integrated into the insect cell genome. ...that is then transformed into a bacterial cell. Transformed cells propagate, are induced to produce your ...

...times), depending on the cell line so old cells should be retired! Conversely, cells younger than the 3rd ...Make sure cells don't stay out too long While you’re preparing things, make sure your cells don’t stay...contaminants. Set up a routine to split cells Don’t be lazy about splitting cells, instead try to form a routine...’s best to split cells before the plates are more than 80-85% confluent because cells like their space...numbers! When starting off with a new cell line, grow the cells, aliquot them into a number of vials, ... Addgene Blog The Challenges of Cell Culture Cloning Mammalian Cells with the Agarose Method Plasmids...lactic acid (a by-product of cellular metabolism that is toxic to the cells), and need to be split very...

...Because each cell can have multiple copies of the transgene that will recombine independently, cells may acquire...expressed. Each cell remains the same color for its entire lifetime and daughter cells retain the same... the fate mapping of cell populations over time. The ability to track single cell dynamics at the organism...expressed in a single cell maintains its color over the entire length of the cell, even axons of up to ... specific cell type. The Confetti mouse was first used to describe the self-renewal of cells in the intestinal...for tracking the fate of Langerhans cells. In the Ubow mouse, cells expressing YFP, CFP, and RFP are not... of CFP++ cells permitted researchers to longitudinally map the turnover of Langerhans cells in the dermis...

...how to affinity purify recombinant antibodies from cell culture supernatant using Protein A or Protein G...after use. Carefully pour the filtered and diluted cell culture supernatant into the Gravitrap column. Collect...

...Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins." Cell Stem Cell 4, no. 6 (2009): ...Jaenisch. "Induced Pluripotent Stem Cells Meet Genome Editing." Cell Stem Cell 18, no. 5 (2016): 573-86. PubMed... express a cocktail of stem cell reprogramming factors and wait for cells to de-differentiate. However...transgene-free and vector-free cells Genome integration Need to sequence cells to verify excision didn’t ...vector-free cells No genomic integration Can be difficult to fully remove virus from cells Plasmid Non-integrating...transduces a wide range of cell types and replicates in the cytoplasm independent of the cell cycle. A challenge...virus which is removed by culturing cells at 38℃, but screening cells for the presence of transgenes is ...

...activated T cells, precluding analyses of T cell differentiation beginning with naïve T cells. The Framework...screens have been used to interrogate T cells, an important immune cell type, in more complex and physiological...naïve T cells. More broadly, this system can be used to perform in vivo screens in other cell types. A...screens to assess T cell responses and clonal dynamics in lymph nodes (LN) and tumor cells. Figure used with...FITS In vitro CRISPR screens in immune cells have identified many putative regulators of immunity. However...Framework for In vivo T cell Screens (FITS) approach (Milling and Markson et al., 2024), developed and deposited...enables efficient knockout (KO) of genes in immune cells; (2) is not immunogenic, a necessity for in vivo...

...mycoplasma contamination in your cell culture or you are about to begin a new cell culture project. If mycoplasmas...present in most of the cell culture facilities, tissue culture labs and every cell culturist has to deal...cost-effective (Sung, 2006). The sensor cell-based method uses specialized cells that can detect the presence of... should be made available for cell culturing and maintenance of the cell lines. Providing such an environment...Photo by Kate Harten DeMaio. Cell lines and reagents Examine your cell line stock for mycoplasma contamination...cryopreserved vials of the cell lines even though the viability of cryopreserved cells decreases with every ...cause mycoplasma infection from one cell line to another. Multiple cell lines should not be stored in a single...

...-B+ cells, and A+B+ cells, but no cells that are A+B-.      Figure 3: It's often easier to identify...population of B cells and dendritic cells (DCs) in the lower middle right. DCs and B cells are HLA-DR+ and...single cells in a population. It can be used to measure protein expression, identify rare cells, or even...looking for cells in a sample that are negative for marker A and positive for marker B, or A-B+ cells. If you...the cell populations as a guide  (Figure 3.) You can see that in our dot plot, we have A-B- cells, A-B...the first step is to separate cells from debris and roughly group the cells by size. This is done using ... and FSC are measured to differentiate cells from debris. Cells are gated, and in the gated population...

...Pluripotent Stem Cells from Blood Cells: An Efficient Protocol Using Serial Plating of Reprogrammed Cells by Centrifugation...human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells, 2013. 31(3): p. 458...possible to reprogram differentiated cells into induced pluripotent stem cells (iPSCs) by the addition of reprogramming... somatic cells that are otherwise difficult to obtain. These can then be used to perform cell therapy ...in vitro. Find stem cell plasmids at Addgene Many sources of adult somatic cells have been probed for...Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic...stem cells from a small amount of human peripheral blood using a combination of activated T cells and ...

...performed on whole-cell extracts from the initial stable cell pool (pool) and from individual cell expansions ... in several human cancers) in A549 cells (a human lung cancer cell line), armed only with viruses obtained...need our A549 cells to express Cas9. In these particular experiments, we generated A549 cells stably expressing...52962-LV, Figure 1). Some cells were then used to generate a monoclonal cell lines by limiting dilution...The rest of the cells were frozen down. Figure 1: Cas9 expression in A549 cells transduced with...blotting was performed on whole-cell extracts from stable pools of A549 cells transduced with Cas9 expressing...A549 cells are relatively easy to transduce. I ended up with too many Cas9 integrations per cell, and ...

...prevent stem cell lines from becoming the new HeLa cells?), genome engineering and stem cells, and the difficulty... landmark Cell paper showing that it was possible to generate induced pluripotent stem cells (iPSCs) capable...forming nearly all cell types in the body from otherwise terminally differentiated cells simply by expressing... Podcast Interview with Stem Cell Researcher Darrell Kotton 2. Stem cell scientists care about patients... happening. Stem cells scientists have certainly not lost site of that. 3. Stem cell science is hard, ...sharing of his OSKM factors have affected the stem cell community – a community largely created due to Yamanaka...understatement. Multiple speakers from top minds in the stem cell field have described the Yamanaka paper as "life...

... a wide variety of cell lines, from HEK to CHO cells to neural and endocrine cells such as AtT-20, PC12...then handled. Additionally, many cell lines, especially the endocrine cell lines we most commonly work with...specific cell line, and its transfection efficiency. We often trypsinize the transfected cells on the day...increased). For some cells, add a drop of serum to aid in outgrowth. Depending on the cell type, we sometimes...growing cells such as HEK and CHO do not seem to require this, while nearly all endocrine cells do. While...difficulty deriving single clones from SH-SY5Y cells, as these cells did not seem to form discrete colonies but... of Medicine. In the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently...

...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...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...mutations influence the disease process. In the Cell Stem Cell paper, for example, we generated hepatocytes...generating the mutant cell lines that one needs to do disease modeling in stem cells. Addgene: Tell me about...Kiran Musunuru's lab in Harvard's Department of Stem Cell and Regenerative Biology is to understand the basis...

...Tagging in Human Cells on the Addgene Blog. Plasmids for Endogenous Gene Tagging in Human Cells Find additional...additional cell line information from the Allen Institute for Cell Science data portal (Link opens in a new...images from the hiPS cell lines gRNA sequences used in the creation of the hiPS cell lines The following...116 mEGFP Proliferating cell nuclear antigen (PCNA) DNA replication foci IPSC cell lines can be ordered ...ordered from the Allen Institute for Cell Science Cell Catalog (Link opens in a new window) . Protocols The ...with this plasmid collection. Single cell passaging of human iPS cells (Link opens in a new window) RNP transfection...for Cell Science has deposited plasmids to generate fluorescently tagged proteins of key cellular structures...

... 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... 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...genetic material from the environment by bacterial cells. In nature, this genetic material often comes from...

...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...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...generate unique cell types. Of the 24 TFs they tested, 22 expressed markers consistent with the cell type that...