...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...

... 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 ...

...added to 150,000 cells resulted in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent...protocol was developed for 293T cells but can be adapted to a variety of target cell lines and selection markers...to determine the percentage of fluorescent cells, these cells can still be used in downstream applications.... Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with...health of the target cell line is critical for obtaining accurate titers. Check the cells for mycoplasma (...regularly Do not over- or under-grow your cells. Thaw a new vial of cells after 20–30 passages. Do not add penicillin...Procedure Seed 75,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute ...

...1,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute 7,000 cells into...protocol was developed for A549 cells but can be adapted to a variety of target cell lines and selection markers...the cells cannot be used for later experiments. Workflow Timeline Day 0: Seed and transduce cells Day ...health of the target cell line is critical for obtaining accurate titers. Check the cells for mycoplasma (...regularly Do not over or under-grow your cells. Thaw a new vial of cells after 20–30 passages. Do not add penicillin...seeding of the cells at the same time as the virus-mediated transduction. For some cell lines, transduction...kill your target cell line needs to be empirically determined. Treat the target cells with a range of ...

...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...

... of HEK293T cells optimized for viral production (AAVpro or Lenti-X), but any HEK293T cell line should...different cell lines and different transfection reagents. Workflow Timeline Day 0: Seed HEK293T cells (or a... health of the cell line is critical for obtaining high levels of virus. HEK293T cells should be split...Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask...8x10 5 cells in a T75 flask in a volume of 15 mL. Do not add antibiotics to the media. Use cells that are...PEI determined. Procedure Seed HEK293T cells at 3.8x10 6 cells per plate in DMEM complete in 10 cm tissue...prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 days after...

...Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest antibody Equipment...10 µL of the cell suspension/trypan blue mix into a cell counting chamber. Load the cell counting chamber...interest in cell culture. This protocol describes how to transfect suspension HEK293 cells with recombinant...Section 1: Seeding cells The day prior to transfecting, seed a 108 mL culture of HEK293 cells at a density ...Pro-Tip Do not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability...transfer 0.5 mL of HEK293 cell suspension into a clean microcentrifuge tube. Pro-Tip Cells settle quickly and...microcentrifuge tube. Vortex the cell suspension. Transfer 10 µL of cell suspension into the microfuge tube...

...commonly referred to as 'competent cells.' Many companies sell competent cells, which come frozen and are prepared...that came with your competent cells. Pro-Tips Commercial competent cells range significantly in their ...SOC media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw...use electro-competent cells. Instead of relying on the heat-shock to cause the cells to take up the DNA,...process by which foreign DNA is introduced into a cell. Transformation of bacteria with plasmids is important... all plasmids (even those designed for mammalian cell expression) carry both a bacterial origin of replication...transformation efficiency. The lowest efficiency cells (usually the least expensive) are fine for transforming...

...such as stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect...and for each cell line. Considerations Before You Start The health of the packaging cell line is critical... viral titer. 293T cells should be split 3 times a week: Monday: Plate 1×10 6 cells in a T75 flask in ...: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Friday: Plate 8×10 5 cells in a T75 flask .... Use cells that are below passage 15 for viral production. Procedure Seed 293T packaging cells at 3.8....8×10 6 cells per plate in DMEM Complete in 10 cm tissue culture plates. Incubate the cells at 37 °C, ...prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after...

...transfected cells. k. Incubate cells at 37°C, 5% CO 2 for 24 hours. Day 4: l. Harvest media from cells and transfer... infect the target cells. Addition of puromycin will allow you to select for cells that stably express... 7×105 HEK-293T cells in 5 mL of media in a 6 cm tissue culture plate. Incubate cells at 37°C, 5% CO 2...Although cells should regularly be passaged in DMEM + 10% FBS with penicillin/streptomycin, cells should... temperature. g. Retrieve HEK-293T cells from incubator. The cells should be 50-80% confluent and in DMEM...you do not want to dislodge the cells from the plate. i. Incubate cells at 37°C, 5% CO 2 for 12-15 hours...antibiotics to the cells and incubate at 37°C, 5% CO 2 for 24 hours. Day 5: n. Harvest media from cells and pool...

...detect antigens in cells. Here we describe the basic steps for fixing and labeling cells in culture with ...2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment Pipette controller Pipette... Seeding cells Place a sterile poly-D-lysine coated coverslip in each well of a 24-well cell culture treated...treated plate. Seed 5 x 10 3 HeLa cells per well. Allow the HeLa cells to grow to the desired density before... the microscope slide with the cell side facing down. Observe the cell labeling on a microscope with appropriate...describes the basic steps for fixing and staining cells in culture with a primary antibody against a target...outlines the steps for fixing and labeling HeLa cells for a target protein using the formaldehyde fixation...

...limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 hours) Cells should be harvested first...electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives include Stbl4 cells or other...Maxipreps rely on incremental, ordered cell lysis. E. coli cells are subject to lysis by freeze-thaw if...Ensure that electrocompetent cells are being used. Chemically competent cells will not provide adequate ...CRISPR pooled plasmid libraries in Escherichia coli cells. Protocols...of a pooled-plasmid library in Escherichia coli cells. Pooled libraries contain tens to millions of different...other ultra-high efficiency electrocompetent cells that are suitable for unstable or recombination-prone...

...Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence into a cell line Isolating...Isolating a Monoclonal Cell Population by Limiting Dilution Generate monoclonal cell lines from a polyclonal...polyclonal pool of stable cells AAV Production in HEK293 Cells Produce adeno-associated virus to deliver your...Extraction Without a Kit Extract RNA from tissues or cells without a commercial kit Plasmid Cloning Name Description...Transfection Introduce plasmid DNA to mammalian cells Lentivirus Production Produce lentivirus with a ... virus from a preparation produced in mammalian cells Watch the Video! AAV Titration by qPCR Using SYBR...Transfection Introduce plasmid DNA to mammalian cells to produce antibodies Antibody Purification Purify...

...1: Seed and transduce cells Day 4: Treat cells with Benzonase and harvest cells Day 4+: ddPCR and analysis...wells with 3,000,000 cells in 13.5 mL of media and 11.1 µg/mL Polybrene. Mix the cell suspension well before...each well. Detach cells by incubating with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete...above, the cells per well is 300,000 and the virus volume refers to the total volume the cells were transduced...used to transduce HEK293T cells. Genomic DNA is extracted from the target cells and assayed for integrated... target cells but can be modified for other targets. Primers and a probe against the cellular ribonuclease... (cations can affect the attachment of adherent cells) Trypsin EDTA (TrypLE, Thermo Fisher, 12605010) ...

... or cells without a kit. The general steps of this protocol include homogenization/lysis of cells or tissues...lyse tissues or cells in Solution D. For tissues: use 1 mL of Solution D per 100 mg of cells. For cultured...cultured cells: use 1 mL of Solution D per 1 X 10 7 cells. Allow sample(s) to sit at room temperature for 5...effectively lyse open the cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4 mL...tissues or cells in TRIzol® or a similar product. For tissues: use 1 mL of TRIzol® per 100 mg of cells. For ...For cultured cells: use 1 mL of TRIzol® per 1 X 10 7 cells. Allow sample(s) to sit at room temperature for... RNA isolation will depend on how effective your cell lysis protocol is. While simple homogenization is...

...a mouse. Procedure Section 1: Lyse cells Centrifuge 5 x 10 6 cells for 5 min at 100 x g . Gently remove...This protocol describes the basic steps for lysing cells, determining total protein concentration in the ...use western blotting to visualize a protein from cells or tissue samples. Equipment Microcentrifuge 0.5...remove supernatant. Resuspend cell pellet in 1 mL of 1X PBS and transfer to a microcentrifuge tube. Centrifuge...100 x g . Carefully remove supernatant. Resuspend cell pellet in appropriate volume of cold lysis buffer...lysis buffer will vary depending on the size of your cell pellet but will generally be between 250–1000 µL...

...investigator to conveniently plate and select transformed cells containing plasmids differing in their resistance...bent to create an “L” shape, and then used like a cell spreader. Several other devices may be used for.... During the incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest. See our...the liquid is mostly absorbed. The spreading of cells can be done in the same way as the antibiotic, using... using either a bent micropipette tip or other cell spreading device that fits the plate. Incubate plates...

...use electro-competent cells instead of the more common chemically-competent cells. The number of bacterial...might need to express YGOI in cultured mammalian cells. The problem is that the only version of full-length... manufacturer’s instructions for your competent cells. For most standard cloning, you can transform 1-...1-2μl of your ligation reaction into competent cells such as DH5alpha or TOP10. If using much less total...colonies, you might want to use higher competency cells. Additionally, if your final product is going to...

... CRISPR Generate genomic deletions in mammalian cells lines using CRISPR/Cas9 Genomic Deletions with CRISPR... virus from a preparation produced in mammalian cells AAV Purification by Iodixanol Gradient Ultracentrifugation... the lab. Blog post: 10 Basic tips for mammalian cell culture Aseptic Technique Aseptic technique is a...fields such as microbiology, molecular biology or cell biology. Learn some of the key strategies of aseptic...

...bacterial cell. Large plasmids usually have a low copy number (approximately one or two copies per cell) and... can be present in large numbers, 50 or more per cell and have a high copy number. High copy number plasmids...increase this to 350 - 400 rpm to obtain a higher cell density. Reference Page Top Index...

...bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria... and you will not need to obtain more competent cells and retransform. Bacteria on an LB agar plate can...

...use electro-competent cells instead of the more common chemically-competent cells. The number of bacterial... manufacturer’s instructions for your competent cells. For most standard cloning, you can transform 1-...1-2μl of your ligation reaction into competent cells such as DH5alpha or TOP10. If using much less total...colonies, you might want to use higher competency cells. Additionally, if your final product is going to...

...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... 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 ...problems with cell lines. Science 2014; 346:1452–1453. Bairoch, A. The Cellosaurus, a Cell-Line Knowledge...problems, like cells lines. A 2014 study in Science found that approximately one-third of cell lines used ...

...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. ...reprogrammed cells based on single-cell gene expression patterns. The lab has also used CellTags for tagging...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...simultaneously tracks clonal history and cell identity at the single-cell level, thus linking ancestral gene...), a self-renewing cell type that can differentiate into hepatic or intestinal cell types. MEFs were reprogrammed...

... 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 ...Animal Cell Culture Guide Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture...working cell bank has been made by a research institution (an excellent practice), or maybe cells have been... 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...

...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... of Stem Cell Research Plasmids . For more information on stem cells, visit the NIH Stem Cell information... reprogramming of human somatic cells to pluripotency. Cell Stem Cell. 2008 Sep 11. 3(3):346-53. Jaenisch...

...complete plasmid can be transformed into bacterial cells for propagation. The majority of ligation reactions...Interpretation Uncut vector - Checks viability of competent cells and verifies the antibiotic resistance of the plasmid...

...Induction of Human Germ Cell Fate from Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94....differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009. 19(...blood cells, and renal epithelial cells from urine are some of the easily isolated somatic cells that ...some of these cell types [13]:  Germ cells [14] Hepatocytes [15] Pancreatic β-cells [16] Intestinal...differentiated cell to a different cell type. While transdifferentiation is suited for switching cell types between... differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 2010. 7(5): p. 618-30. PubMed...et al., Reprogramming of T cells from human peripheral blood. Cell Stem Cell, 2010. 7(1): p. 15-9. PubMed...

...Pharmacological Sciences, a Cell Press reviews journal. Stem cells are special types of cells that can develop ...specialized cell types [2]. We general speak of 4 levels of stem cell potency: Totipotency: When a cell has ...pluripotent cells can be thought of as descendants of totipotent cells. An example of pluripotent cells is embryonic... into myogenic precursor cells [5]. Classification of stem cells Stem cells are classified into two broad...Embryonic stem cells ESCs are pluripotent stem cells that are able to form all adult cell types and are... Adult stem cells (ASCs) Also known as somatic stem cells, these multipotent stem cells are located ... to maintain cell numbers or replenish dying cells. For example, hematopoietic stem cells found in the...

...plot of CD3 (T cell marker) versus CD19 (B cell marker) to separate the B cell and T cell into distinct...separate the B cell population into progenitor B cells, immature B cells, plasma cells or others.   ...determining cell health, analyzing cell cycle stages, and detecting specific populations of cells in a complex.... As cells pass through the flow cell the size of the sample stream is reduced forcing the cells to line... photons varies from cell to cell depending on the level of fluorescence. A cell expressing high levels...studies B cells, might then gate the B cell population and create a histogram of CD19-containing cells versus...CD3 versus CD19 to separate B cells, T cells and natural killer (NK) cells.   Tips for flow cytometry...

...out all CD8+ T cells from a mix of CD8+ T cells, CD4+ T cells, B cells, and dendritic cells. In this case...dumped”. From left to right: B cell, CD4+ T cell, dendritic cell, CD8+ T cell. Created using biorender.com...amines on the cell surface, which yields a much weaker signal on live cells compared to dead cells. As a result...in flow cytometry are cell proliferation and cell viability. Unlike intracellular labeling, dye staining...be done on live cells. For cellular proliferation, esterified dyes that can enter cells freely are typically...comes to labeling cells for flow cytometric analysis, the most common method is a cell surface label, where...through the cell membrane freely, while others require a permeabilized membrane to enter cells. Depending...

... competent cells (protocol)  Transformation of electrocompetent cells (protocol)  Cells prepared using...to make your own competent cells It’s easy to make your own competent cells. If you need a high efficiency...purchasing competent cells. However, for most everyday cloning, homemade competent cells will work just fine...Preparing chemically competent cells (protocol)  Preparing electrocompetent cells (protocol)  Untergasser...of chemically competent cells (protocol) Preparation of electrocompetent cells (protocol) At Addgene...Set  from Zymo Research to make competent cells because cells prepared using this kit can be transformed...then wash and resuspend the cells in the provided buffers. The competent cells are then aliquoted and stored...

...Many cell types can be found in the tumor microenvironment (tumor cells, immune cells, blood cells and ...about 53% of my cells are dendritic cells and ~13% are CD8+ T cells. Around 34% of the cells are neither ...neither of the two cell types (likely B cells and others). Almost no cells stain positively for both markers...measure your cells of interest. In many experiments you’ll be working with a heterogeneous cell population...processed piece of tissue, where cells are present that vary in cell type, size, and marker proteins. ...analyzing a homogenous cell population in culture, it is normal to observe diversity in cell size, marker expression...illuminates the passing cell with a laser and detects the light scattered by that cell at a low angle (FSC...

...labeling T cells, B cells, and antigen presenting cells, scientists have been able to decypher cellular dynamics...tracked by cell sorter giving immunologists the ability to count cells and statistically analyze cell movements...fast T cells could jump from one antigen presenting cell to another. Indeed, we now know that T cells are...2016)? How do dendritic cells migrate to lymph nodes to activate T cells (Celli et al 2008, Kitano et al...In complex metazoans, rapid cell division and large scale cell mobility are essential processes during.... a basal progenitor cell migrating to the epithelium). At this stage, only cells from the immune system...to directly follow immune cells in lymphoid organs. The development of cell sorters together with the ...

...activity, and consists of other cell types, especially immune cells and cells of epithelial origin. Suppressing...turns off T cell function upon interaction with APCs. Furthermore, tumor cells alter their cell-surface proteome...molecules needed for T cell effector function. Cancer cells also produce programmed cell death ligand (PD-L1... activity of CTLs and NK cells. These cells would normally kill cancer cells but are directed not to through...to use whole cell therapeutics consisting of various types of immune cells. Dendritic cells (DCs) are particularly... intravenously. B cells and T cells activated ex vivo are also contestants for cell-based immunotherapy... include: Cell heterogeneity Tumors are composed of a wide variety of cell types. This cell heterogeneity...

...transfected cells may be considered an entirely new cell line from that of the original parental cells. Positive...cultured mammalian cells. Of course, these mammalian vectors must be compatible with the cell type they are...bacterial cells); however, the techniques and reagents vary. Plasmid transfection into mammalian cells is fairly...expression levels in the cells. Ideally, the chosen reporter is unique to the cell, is expressed from the...components expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr...mammalian cells To achieve stable transfection, there should be a selective pressure to force cells to incorporate...(basically growing cells in various concentrations of the selection reagent). Cells should die within ...

... hiPS cell lines generated using these plasmids, please visit the Cell Catalog on the Allen Cell Explorer...Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen... in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving..., and the stem cell lines we’ve made using them are available as part of the Allen Cell Collection at ... and images generated from these cell lines can be found in the Cell Catalog on our data portal. The plasmids...transfect the cells, these large regions of homology enable us to effectively utilize the host cell’s inherent...

... 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...

...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...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... your cells can catch a cold—or worse! HEK-293T and its derivatives are immortalized, but cell health...