...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...