...Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day 7 (am): Harvest cells Equipment... 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...media and cells. Centrifuge at 3900 rpm for 20 min at 4 °C to pellet the cells. Keep the cell pellet on... Protocols AAV Production in HEK293T Cells AAV Production in HEK293T Cells You may also...

... the cell media. Cell death by some cells in the culture may adversely affect the surviving cells in the...media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute...conditions for the surviving cells. Even in the absence of cell death, the cell media should be changed every...monoclonal cell lines from expansion of individual A549 cells stably expressing Cas9. A549 cells were transduced...Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus You ...experiments in a stable cell line, as opposed to transiently-transfected cells, increases reproducibility...a stable cell culture after transduction. Performing antibiotic selection on transduced cells enables ...

...of a 5 cell/mL solution. Calculate the total cells needed: Total cells needed: 10 mL × 5 cells/mL = 50...generate a monoclonal cell line from a polyclonal pool of stable cells. Transducing cells with lentivirus results...polyclonal cell pool. Generating a monoclonal cell line by limiting dilution results in cell populations...Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day 14–30: Analyze...Isolating a Monoclonal Cell Population by Limiting Dilution Isolate cells from the stable cell pool by trypsinization...through a 40 µm cell strainer mesh. Count the cell concentration in this homogenized cell solution using...hemocytometer or other cell counter. Pro-Tip Prepare a relatively dilute cell solution (<10 6 cells/mL) before counting...

...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...

... 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...protocol is for suspension cells. Adherent cells can either grow as individual cells in 96-well flat bottom...Deletions in Mammalian Cell Lines CRISPR: Protocol for Genomic Deletions in Mammalian Cell Lines Other Addgene...detail for murine erythroleukemia (MEL) cells, a suspension cell line. The culture medium in all steps ...numerous cell types using preferred culture conditions and transfection strategies for each cell type. While...Ensure there are 2 x 10 6 cells per CRISPR pair. Resuspend 2 x 10 6 cells in 100 μl of electroporation solution...

...Consortium plasmids that encode the extracellular regions of cell surface receptor and secreted proteins...expression plasmids that encode the extracellular regions of cell surface receptor and secreted proteins...screens or cellular assays to identify novel interactions or pathways that are important in cellular recognition...AVEXIS (for Avidity-based extracellular interaction screen) in which the extracellular regions of receptor ...and Wright GJ Molecular & cellular proteomics 2010 Dec; 9(12):2654-65. A cell surface interaction network... Plasmid Collections EXPRESs Plasmids EXtracellular Plasmid RESource (EXPRESs) Consortium You may...The inserts that encode the whole ectodomains of cell surface receptors are flanked by two 8 bp cutting...

...give the cell its shape, organize cellular organelles, and are necessary for movement and cell division...between animal cells. They are important for holding cell structures together and reducing cell stress. There... General Structures The cell contains a variety of organelles and subcellular structures each with their...example, houses the cell’s genetic material. The mitochondria produces the energy cells required for chemical...kappa-myc-dL5-2xG4S-TMst Cell surface (mammalian) PDGFR-derived dL5 FAP; mCerulean3 Marcel Bruchez 45944 pTDpelB-C_sfYFPTwinStrep... on cell cycle) Centrin-2 EGFP Erich Nigg 41151 pEGFP Cep170 C-term Centrioles (dependent on cell cycle...Fluorescent Protein Collection . Return to top Cell Junctions Cell junctions are responsible for forming connections...

...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 ...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 ...visualization of proteins inside living or fixed cells. The plasmids associated with this technology are...

...in which the cell line was described. Using Cellosaurus Cellosaurus is searchable by cell line name (recommended...resource called Cellosaurus, a growing cell line information database describing nearly 145,000 cell lines. Cellosaurus... Cellosaurus Cellosaurus seeks to identify and describe all cell lines used in research, using four methods... 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...

... horizontal cells, and Cre-dependent-GFP expresses in horizontal cells and ganglion cells, only the horizontal... 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 target a cell class in mice is not a good predictor for targeting the same cell class in NHPs (...

...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...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... ability to propagate viruses in cell culture; if there were no cells to infect, there would be no viable...

...should start celebrating papers and grants too with pizza or pub.” The Cepko Lab celebrates any fellowships.... Once goals are reached, how do science labs celebrate accomplishments and their team's hard work? Do...team and manage work-life balance. How do you celebrate your lab’s achievements? Tom Ellis highlights ...to the pub. Dr. Ellis adds “we probably don’t celebrate enough... I’ll ask my team at our next group meeting...dinner and champagne in the lab. The Church Lab celebrates successful moments in the lab with parties in...professional growth along the way. Take time to celebrate the lab’s achievements and enjoy time with friends...to have fun outside the lab! How does your lab celebrate each other's accomplishments? Any tips on making...

...Addgene has so much to celebrate! As we enter this new growth phase of the organization and the expansion...teams continue to keep up with the demand with excellence and dedication. We don’t have worry or wonder...right? Upgrades to the internal LIMS pages and excellent work on our external website appearance and functionality...support. The Addgene management team leads with excellence. They are one of the main reasons I have so few...Addgenies. I never worry about their dedication, excellent intentions, or incredible drive to succeed.  Thanks...

...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...

... 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...