...Pre-implantation Blastocyst Cell Types from Fibroblasts. Cell Stem Cell. 2019 Jun 6. Buganim Glial Cells Retinal Ganglion... Plasmids Stem Cell Research Plasmids Stem cells are proliferative, unspecialized cells with the capacity...different cell types in the body during development. Unlike other cell types, each stem cell has the potential...generate different cell lineages. Pluripotent stem cells, such as embryonic cells, can potentially give...any other cell type in the body. Multipotent stem cells, such as adult (or somatic) stem cells, can differentiate...disease-specific cells. For this reason, iPSCs have become an excellent unlimited stem cell source for therapeutic... of Stem Cell Research Plasmids . For more information on stem cells, visit the NIH Stem Cell information...

... Depositor Collections Cell Migration Consortium (CMC) Plasmids Cell Migration Consortium (CMC) Plasmids...collaboration between the Cell Migration Consortium and Addgene. Member laboratories of the Cell Migration Consortium...This resource is a collaboration between the Cell Migration Consortium (CMC) and Addgene, which highlights...Consortium have deposited plasmids useful for cell migration research at Addgene. CMC investigators who have...MIT Rick Horwitz , University of Virginia Denise Montell , University of California, Santa Barbara Martin...

...obtain approximately 30 cells per 96-well plate. Include 100 μl per well of cell culture media. For the...is for suspension cells. Adherent cells can either grow as individual cells in 96-well flat bottom plate... While MEL cells are suspension cells, instructions for adherent cells have also been included. Ensure... Fluorescence Activated Cell Sorting (FACS) of Transfected Cells Prepare cells for FACS by filtering them...limiting dilution at 30 cells per 96-well round-bottom plate. Optimize plating the cell type used at limiting...parental and bulk sorted cells by resuspending parental and bulk cell pellets in 50 μl of DNA extraction...suspension cells, transfer all clones to a single 96-well plate that already contains 50 μl cell culture ...

...give the cell its shape, organize cellular organelles, and are necessary for movement and cell division...these well-characterized proteins. General Structures The cell contains a variety of organelles and subcellular...between animal cells. They are important for holding cell structures together and reducing cell stress. There...Collections Fluorescent Proteins Organelle Markers Fluorescent Proteins: Subcellular Localization More Fluorescent...proteins that are targeted to various organelles or subcellular structures. You should fuse your protein...example, houses the cell’s genetic material. The mitochondria produces the energy cells required for chemical...the table below to find markers of general cellular organelles and structures. ID Plasmid Structure Targeting...

...for Cell Science has deposited plasmids to generate fluorescently tagged proteins of key cellular structures... Protein Guide Subcellular Localization Plasmids Protocols The Allen Institute for Cell Science has produced...fluorescently tagged proteins of key cellular structures in human cell lines of their choice. For a full ...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...plasmids can be used to create cell lines with endogenously-tagged cellular structures. Addgene ID Plasmid...

... sub-cellular localization (membrane, nucleus, etc.) are also available Non-toxic to living cells To learn...repair protein, and is suitable for both cell-permeant and non-cell-permeant proteins. CLIP-tag CLIP-tag ... (CoA). These tags are particularly well suited for labeling cell-surface proteins, and are useful for...Control for cell surface localization 101131 pACP-GPI Control Plasmid ACP/MCP-tag Control for cell surface...visualization of proteins inside cells. Depositor... Depositor Collections New England Biolabs Cell-Imaging Plasmid Collection...Collection New England Biolabs Cell-Imaging Plasmid Collection Technology Plasmids Supplemental Information ...

...media and cells. Centrifuge at 3900 rpm for 20 min at 4 °C to pellet the cells. Keep the cell pellet on ice...Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day 7 (am): Harvest cells Equipment...resuspend each pellet completely. Combine resuspended pellets and keep on ice. Process the cell pellet from above... HEK293T cells is critical for optimal AAV yield. Do not overgrow your cells. Pass the cells twice a week...multiple times to obtain a single cell suspension (no clumps of cells). Pool cells from 2 x T-175 flasks. Adjust...obtain a single cell suspension (no clumps). Count cells using a hemocytometer or cell counter. Seed 350...Harvest cells and media by tapping the sides of the CS5. Cells should detach easily. Transfer cells and media...

...control well). Perform a "reverse transduction" by seeding 50,000 cells into each well of the 6-well dish.... Aliquot 1 mL of cell suspension (i.e., 50,000 cells) into each well of the 6-well dish. This brings ... the cell media. Cell death by some cells in the culture may adversely affect the surviving cells in the...single viral dilution to each well (each well gets one dilution, so a 6-well plate will hold 5 dilutions...media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute...stable cell pool. Observe the dish every day to ensure that the cells in the untransduced well (0 µL lentivirus...conditions for the surviving cells. Even in the absence of cell death, the cell media should be changed every...

...0.5 cells/well. Seeding an average of 0.5 cells/well ensures that some wells receive a single cell, while...Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day 14–30: Analyze...homogenized cell solution. Homogenized cell solution concentration: 4 × 10 5 cells/mL To seed one 96-well plate...of a 5 cell/mL solution. Calculate the total cells needed: Total cells needed: 10 mL × 5 cells/mL = 50...any well receives more than one cell. Pro-Tip In each 96-well plate, seed one of the corner wells with...within each well, especially in the corner of the well as cells tend to settle there. Once the cells have expanded...generate a monoclonal cell line from a polyclonal pool of stable cells. Transducing cells with lentivirus results...

...nearly 145,000 cell lines. Cellosaurus Cellosaurus seeks to identify and describe all cell lines used in...in which the cell line was described. Using Cellosaurus Cellosaurus is searchable by cell line name (recommended...reached out to us to tell us about a resource called Cellosaurus, a growing cell line information database... related cell lines when annotating a cell line Community submissions via email: cellosaurus@sib.swiss...with cell line catalogs and collections. Importantly, Cellosaurus also identifies problematic cell lines...HeLa cells. Image courtesy of Josef Reischig.  Contributing to Cellosaurus Generated a new cell? Or ... about a cell line you’ve used? Submit your manuscript or data to Cellosaurus via cellosaurus@sib.swiss.This...

...Tracking lineage and cell identity at the single-cell level with CellTag CellTagging is a lentiviral based...individual cells. Each cell is labeled with 3-4 tags to increase the diversity of the CellTag library. ....    Find CellTag libraries here!  Applications of CellTag The Morris Lab developed CellTag to track the...reprogrammed cells based on single-cell gene expression patterns. The lab has also used CellTags for tagging...et al., 2019). Key takeaways of CellTag CellTag is a combinatory cell-indexing tool that combines scRNA-seq...describes the process where differentiated cells are turned into a cell type of choice while bypassing the intermediate...majority of cells failing to develop the desired identity. The development of single cell technologies...

...organs, cells comprise compartments and structures, called organelles. Take a sneak peak inside a cell with...vesicles via the secretory pathway to their subcellular or extracellular destination. At the end of their life...which are known to be a part of an organelle or involved in intracellular transport pathways, it is possible...proteins, however, in this case the cell needs to be fixed and the cell membrane permeabilized in order for... to reach their intracellular targets. Therefore, it can be beneficial to employ well-characterized marker...for labeling your subcellular structure of interest in the Allen Institute for Cell Science Plasmid Collection...and selected subcellular marker proteins, it’s possible to get an idea about the subcellular structures ...

...Chapter 9: Using stem cells as in-vitro models of disease. How Darrell is using stem cells from patients to... School. Darrell’s research focuses on the lungs, and, among other projects, using stem cells to develop...about Darrell, his research, and his focus on open science. Listen to the interview with Darrell Kotton...Introduction to Darrell Kotton and the Boston University Center for Regenerative Medicine. Darrell describes...him as a praticing physician and stem cell researcher. Darrell's ultimate goal - to develop therapies ...Kotton Lab. One of Darrell's favorite projects - differentiating surfactant producing cells that can be used...engineered cells in therapy. The lungs are difficult, but other tissues like the retina have cell-based therapies...

... horizontal cells, and Cre-dependent-GFP expresses in horizontal cells and ganglion cells, only the horizontal...improve cell-type specificity of AAV, work has focused on modifying the outside protein shell, or capsid... in retinal cells. To do this, the group developed a new strategy for finding retinal cell-type specific...and high cell-type specificity.  Strategy #3 (ProC): Create promoters by repeating mouse cell type specific... to test for cell type-specific expression of GFP. Half (113) expressed in retinal cells four weeks post-injection...targeted expression in specific cell types (ex: rods or cones) or cell classes (ex: photoreceptors which...occurred in one type of cell More than 50% of expression occurred in one type of cell, with the remaining ...

...ability to tell stories a similarly crucial skill for career advancement. Scientists do and should tell stories...interviews. They even tell short stories during weekly lab meetings. When scientists tell good stories, they...story short, telling a good story is something that can be learned, and will serve you well in your career...career. Story Telling at work Don’t believe me? Here’s an example of storytelling to answer an informational...be memorable. Work out what story you want to tell; tell it; stop. Many thanks to our guest blogger Jack...The part of my job I most enjoy is building and telling interesting stories — whether I’m working with ...when talking to friends or dates or colleagues we tell tales about parts of our lives that tend to have...

...more generally “How am I doing? Can you tell me what’s going well and what I could improve on?” Having ...have time to answer outstanding questions you have, well before experimental crunch time. Don’t: Wait to...at times! What makes it rewarding is when you can tell a trainee is understanding and things are ’clicking...

...quality work in cell culture. The problems with cell lines Simply put, the majority of cell line problems...HeLa cells, the first immortalized human cell line. He proved this by examining whether the cell lines...Cancer Cells, Marker-independent identification of glioma-initiating cells. The International Cell Line ...working cell bank has been made by a research institution (an excellent practice), or maybe cells have been...passage number, cell line, and date, which you complete each time you passage the cells (as well as recording...Animal Cell Culture Guide Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture... Outreach Scientist Nick Amiss. Cell culture in the present day Cell culture is a widespread tool used...

... to microbes by using non-invasive methods for cellular imaging with ultrasound. In his research he applies... take home message was that being creative and telling a story makes scientific topics both interesting...

...pursuit of achieving knowledge. Or, as we in the storytelling business like to say, science is all about navigating...all science communication, what you are doing is telling a (nonfiction) story. For there to be a story, ... journey? The same thing applies for science storytelling. You can introduce your results right at the...the science into. There are almost limitless storytelling options, and they all can be effective in the...circumstances.   However, in the midst of all these storytelling devices, we sometimes forget the most important...

...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...RJ, Rehmann H, Geijsen N. Cell. 2015 Apr 23;161(3):674-690. doi:10.1016/j.cell.2015.03.028. PubMed. Find...

...times), depending on the cell line so old cells should be retired! Conversely, cells younger than the 3rd ... Here are some tips for cell culture that will hopefully help you keep a well organized lab and contamination...hands! You should additionally remove all jewellery as well as that watch you’re wearing before you start...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...results. Most cell culture media contain the pH indicator phenol red (pH range 6.0 (yellow) - 8.0 (red)...

...into 96-well plates, is extremely time- and resource-consumptive, since only wells with one cell can give...add a drop of serum to each well in the 48-well plate; rapidly growing cells such as HEK and CHO do not...while nearly all endocrine cells do. While not all of the wells in the 48-well plate will grow up, most ...Biosciences) to transition from a 48-well cell plate to a 96-well assay plate. We generally freeze at ... 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...and glucose. If we circled well-separated clones containing more than 50 cells (visible to the naked eye...

...psMEK1 in these same cells by 24-48 hours exposure to blue light also induced tail swelling in ~75% of worms...of Multiple Kinase Activities in Live Single Cells." Cell 157, no. 7 (2014): 1724-734. PubMed PMID: 24949979...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to ... dimeric Dronpa is less prone to aggregation in cells compared to the tetrameric Dronpa it was derived...inhibitors, psMEK1 and ERK KTR-mRuby2 expressing cells were incubated with an inhibitor of choice, exposed...inactivation, and then decreases. Treatment of the cells with a PP1/PP2A inhibitor leads to maintenance of... use rectal bacterial infection to induce tail swelling in C. elegans, but activation of Raf-MEK-ERK signaling...

...and analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is...cytosol and organelles to the vacuole/lysosome for degradation and recycling. The key to Rosella’s autophagy-sensing...with the vacuole or lysosome. Rosella fluorescent properties Rosella is a dual color-emission biosensor...portion of Rosella: it will emit red fluorescence regardless of its localization in the cell. SEP’s pH ...color fluorescence Rosella emits based on its localization. Table 1: Summary of Rosella’s excitation and ...fluorescent properties of Rosella.   Table 2: Effect of Localization on Rosella’s fluorescence emission. ...Lysosome  ~4.8  Red    Using Rosella to study autophagy in yeast Rosella is targeted to the cytosol when...

...the SeqWell website to learn more about our plexWell kits and services! Many thanks to Joe Mellor, our...our guest writer from seqWell Inc. Joe Mellor is the Founder and CEO of seqWell, Inc. He is a sequencing... contributed by guest blogger Joe Mellor, Founder and CEO of seqWell Inc. Plasmids and PCR products are...DNA-barcoding step that uniquely labels DNA from each well of a 96-well plate. Then, samples from several plates are... to 1000s of samples individually, plexWell™ reduces every 96 well plate to a single tube after the first...across samples, NGS data obtained via plexWell™ libraries is well suited to large-scale de novo assemblies...from plexWell™ NGS data. Once Addgene produces plates of plasmid DNA, they are shipped to seqWell, where...

...Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen...stem cell lines we’ve made using them are available as part of the Allen Cell Collection at Coriell. More... hiPS cell lines generated using these plasmids, please visit the Cell Catalog on the Allen Cell Explorer... tagging to elucidate cellular structures. Overexpression methods flood the cell with protein, which can... in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving... and images generated from these cell lines can be found in the Cell Catalog on our data portal. The plasmids...

...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...

...varying across cell types. Variance can be caused by cell cycle rates; rapidly diving cells will have higher... of your cells. Enrich S-phase cells HR operates predominantly in S and G2 phase of the cell cycle, when...HDR events, ensure cells are actively cycling through the cell cycle stages. If cells are overly confluent...way to make a knock-in cell line is to utilize the built-in repair pathway cells already have for repairing...than quiescent cells. It is important to know that HR is often mutated in cancer cells, specifically in...keeping an eye on those cells! To take this one step further, you can inhibit cell cycle regulatory proteins...negative outcomes on viability if cells are arrested too long. Prolonged cell cycle arrest can lead to apoptosis...

...prevent stem cell lines from becoming the new HeLa cells?), genome engineering and stem cells, and the difficulty... Podcast Interview with Stem Cell Researcher Darrell Kotton 2. Stem cell scientists care about patients... 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... 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...

... do so, their T-cells were grown in vitro, their mutations corrected, and the T-cells were transferred...functioning T cell repertoire (1). In this example, it was possible to transduce the T-cells outside the...a wide range of cell types including but not limited to muscle, liver, and brain cells. There are a multitude...chain (c) and is characterized by a lack of immune cell development and function. To date, gene therapy ...that can deliver gene therapies directly to the cells that need them. Adeno-associated virus (AAV) – based...delivery of nucleic acid cargo (DNA or RNA) to target cells has been challenging. A variety of vector delivery... delivery vehicles with great safety profiles as well as other benefits. What makes AAV vectors great...