Jan 18, 2007 our protocol describes the use of stably transfected mammalian cells that are propagated in monolayer culture and are readily adaptable for suspension growth of 293t cells in spinner cultures as well. This includes introducing new genes to study or leverage their function, as well as using other constructs to indirectly influence endogenous gene expression or other. Introduction follow the protocol below to thaw freestyle 293f cells to initiate cell culture. New and improved secondgeneration aggrewell plates are.
Hek 293293t cells are derived from human embryonic kidney. Procedural guidelines subculture the freestyle 293f cells a minimum of three times to allow them to recover from thawing before using them in transfection experiments. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Cell culture basics handbook thermo fisher scientific. Protocol for freezing cells when you have started a new cell line it is a good policy to freeze down a good portion of the cells for use at a later date. The hek293t cell line is supplied as a single vial containing 1 x 106 frozen cells in. Hek 293 cells grown for several days in standard tissue culture medium. Protocol for thawing cryopreserved tzmbl and 293t17 cells. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Hek293 is a cell line derived from human embryonic kidney cells grown in tissue culture. You may notice a more contracted morphology in the transfected 293tft cells than in nontransfected counterparts, as well as the presence of small black dna precipitate particles in the open spaces of the plate fig. Human embryonic kidney 293 cells, also often referred to as hek 293, hek293, 293 cells, or less precisely as hek cells, are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture. This protocol assumes that the cells are being cultured in t150 flasks. Approximately 1824 hours before transfection, plate cells in 2.
Aggrewell plates bring an easy, standardized approach to the generation of cell aggregates, including embryoid bodies ebs and spheroids. Drug screening and phenotypic analysis in a microwellbased 3d cell culture system. I have a protocol for the transfection of hek293 cells that use mem medium but i am using hek293t cells. Murine microglial cell line bv2 cells kindly provided by katerina akassoglou and macrophage raw264. Antibiotic resistance 293ft cells stably express the neomycin resistance gene from pcmvsport6tag. Flasks should be incubated at 37c in 5% co2 and hek293 cell doubling time is approximately 34 hours. Wipe the surface with 70% ethanol and drop the cell suspension gently into a centrifuge tube containing 9. The freestyle 293f cell line is supplied in a vial containing 1 ml of cells at 1 x 107 viable cellsml in 90% freestyle 293 expression medium and 10% dmso. Add prepared transfection complexes from step 3 or 4 7. Hek293 cells human embryonal kidney cells hek293, atcc. Cell monolayers are disrupted and removed by treatment with trypsinedta at confluency. Using a 10 ml pipet, flush the loosely attached 293t cells off the culture dish with icecold pbs. Explore these workflows to learn more about the other products we offer to support each research area.
Add 6l of lipofectamine2000 from biostores and 100 l of. Hek293 cells should be grown in a complete sfmii growth medium supplemented with 4 mm lglutamine. Using this protocol your cells will be ready to split again in about 34 days. Repeatedly suck suspension into pipet then expel into the flask, thus breaking up cell chains and groups. You may notice a more contracted morphology in the transfected 293t ft cells than in nontransfected counterparts, as well as the presence of small black dna precipitate particles in the open spaces of the plate fig. Hek293t cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due.
Hek 293 cells should be grown in a monolayer, preferably in plastic petri. Starting lentix 293t cell line cultures from frozen stock. Growth and maintenance of the 293ft cell line thermo fisher. The transformation resulted in the incorporation of approximately 4. Our protocol describes the use of stably transfected mammalian cells that are propagated in monolayer culture and are readily adaptable for suspension growth of.
A summarized submerged organoid culture and passaging protocol is shown in the figure below. Protocol 2 producing lentivirus in hek293t cells using a. Prewarm 50ml of optimum stored in cold room at 4c 10min. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Trypsinize cells from the desired number of flasks or plates. Calcium phosphate transfection of 293t cells 2 transfection 1. The dna to be expressed is cloned into the pmt2t or related. Basic protocol 3 describes the assessment of genome editing ef. Each method is comprised of a commerciallyavailable base medium to which standard cell culture supplements such as hifbs and lglutamine were added according to the manufacturers adaptation protocol. Protocol for hela cell culture and plasmid transfection av02hela cell culture 1. The a549 cell line grows easily and cell count doubling time is typically 2440 hours. T75 cell culture flask or 60 ml petri dish for smaller culture. Hek 293 cells are straightforward to grow in culture and to transfect.
Thaw freestyle 293f cells directly into the freestyle 293 expression medium supplied. Transient plasmid dna transfection protocol per well of a 6well plate a. Typically, these experiments involve transfecting in a gene or combination of genes of interest, and then analyzing the expressed protein. This particular line was initiated by the transformation and culturing of normal hek cells with sheared adenovirus 5 dna. Protocol for virus titer by flow cytometry day 1, prepare hek293 t cells 1. For example, if you thaw a vial of cos cells to carry the cell line. Thaw the frozen vial in a 37c water bath for 12 minutes. Thaw the vial as soon as possible upon receipt to ensure its viability. Hek293 cell culture and transfection hek293 cell line. We strive to make cell culture research more efficient and convenient for researchers by offering outstanding quality, consistency, and value.
It is easy to grow and transfect and have been widely used for cell biology research and also used by the biotechnology industry to. The 293t cell line, originally referred as 293tsa1609neo, is a highly transfectable derivative of human embryonic kidney 293 cells, and contains the sv40 tantigen. For example, if you thaw a vial of cos cells to carry the cell line you will eventually split the cells into 1015 plates. It gives high titers when used to produce retroviruses. Animal cell culture guide tips and techniques for continuous cell lines 1. Protocol 2 producing lentivirus in hek293t cells using a 2. Nhbe a, c and e or hek 293t b, d and f cells were transfected with an empty vector control, or a flagwt rigi expression vector rigi. Determine viable and total cell counts and calculate the volume of freezing medium required to yield a final cell density of. Why are hek293 cells grown in mem but hek293t cells are. To a t75 flask, add 2 ml of trypsinedta to the flask and observe for cell layer detachment under an inverted microscope. Freezing the lentix 293t cell line once the culture has been started and the cells are growing normally, you should prepare frozen aliquots to. Ebs and spheroids generated using aggrewell plates are consistent in size and shape, and are uniform within and between experiments.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Characterizing cancer cells with cancer stem celllike. Incubate the cells at 37 oc5% co 2 overnight the cells will become about 5060% confluence on next day. Culture of hek 293293t cells charles yin last updated. Trypsinedta treatment for disruption of 293t17 and 293sgnt1 cell monolayers note 3. Protocol for thawing cryopreserved tzmbl and 293t17 cells august 2018 i. The protocol does not require removal of serum or culture medium and does not. Hek293 cells should not be used for nucleofection after passage number 20. An optimized protocol for protein purification in cultured. Primary cell culture guide tips and techniques for culturing primary cells 784 kb. We found that culturing 293t cells in a t175 flask allowed us to produce a large amount of virus in a single vessel and to feed 293t cells more gently. Approximately 1824 hours before transfection, plate cells in. After over a decade of seeding smaller scale 293t fresh from atcc i can say with confidence that invitrogen hela based chart never really worked out for me to obtain 7080% for 293t, the. Cryopreserve freestyle 293f cells freestyle 293f cells can be frozen directly in freestyle 293 expression medium.
Growth and maintenance of the 293ft cell line thermo. The human hek293t cell line is a derivative of the hek293 cell line, a human embryonic. Hek 293 293t cells are derived from human embryonic kidney transformed with adenovirus 5 dna hek cells are easy to culture and transfect, and contain the sv40 large tantigen, which allows for the. This product is designed for use in the following research areas as part of the highlighted workflow stages. Cell culture media for suspension cells include excell 293 safc biosciences inc. Culture conditions the cells should be preferably passaged 23 days. Solving the aggregation problem of human embryonic kidney. Transfection methods for cell culture transfection is generally defined as the process of introducing dna, rna or proteins into cells to influence their genotype or phenotype. Renew medium 23 times a week, and keep cell count between 1 x 10 5 and 3 x 10 5 cellsml.
The cells were then either mock treated or infected. Protocol for trypsinedta treatment for disruption of cell. Basic protocol 2 describes the transfection of hipscs, while the alternate protocol outlines the same process for hek293 cells. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Thawing the cryopreserved cells properly is crucial to ensure the viability and functionality of the cells throughout usage in the assays. Prepare an extra plate for cell counting on next day. Propagation of tlahek293t ordering information tlahek293t.
Jan 19, 2011 the in vitro expression and electrophysiological recording of recombinant voltagegated ion channels in cultured human embryonic kidney cells hek 293t is a ubiquitous research strategy. The in vitro expression and electrophysiological recording of recombinant voltagegated ion channels in cultured human embryonic kidney cells hek293t is a ubiquitous research strategy. Protocol culture conditions the cells should be preferably passaged 23 days before before nucleofection nucleofection. Jul 08, 2010 293t cells form spheres in stem cell promoting culture that are enriched in cancer stem cell markers. Freestyle 293 expsys 250439c thermo fisher scientific. Finally, basic protocol 4 describes a method to isolate monoclonal hipsc colonies with desired genotype. Cells should be nucleofected after reaching 8090% confluency.
Assay for phenotype or target gene expression 48 72. Optimized transfection strategy for expression and. Upon thawing immediately place cells in a 25 cm2 cell culture flask with 10 ml of pre warmed. Hek 293 cell, the human embryonic kidney 293 cells was originally derived from human embryonic kidney cells grown in tissue culture. Protocol for hela cell culture and plasmid transfection av. I am growing the hek293t cells in mem currently and they are doing all right, but i wonder. When using our spinfection protocol there is no need to concentrate the virus. Hek293 are rounded cells that grow in suspension in cell culture, although initially they were an adherent cell line.
They are also known, more informally, as hek cells. Freeze freestyle 293f cells at a final density of 1. The widespread use of this cell line is due to its transfectability by the various techniques, including calcium phosphate method. Trypsinedta treatment for disruption of 293t 17 and 293sgnt1 cell monolayers note 3. Crisprcas9directed genome editing of cultured cells. Volumes used in this protocol are for 75 cm2 flasks. This method removes the cyropreservative and can be beneficial when resuspending in. Culture 293t cells in complete dmem and 293tcep4sivtat cells in. Hek293 cells methods, protocols and troubleshootings. Do not allow cell concentration to exceed 1 x 10 6 cellsml.
Commitment to quality we understand the importance of lottolot. Digest 293t cells at log growth phase and seed the cells into 6well plate with 5x105 cellswell. Protocol for subculturing hek293a7 cell line protocol. This cell line is competent to replicate vectors carrying the sv40 region of replication.
Culture the desired quantity of 293ft cells to 7090% confluency. Perform all steps in a tissue culture hood under sterile conditions. Freezing the lentix 293t cell line once the culture has been started and the cells are growing normally, you should prepare frozen aliquots to provide a renewable source of cells. Aspirate unused cell solution and discard used cultureware. Fugene 6 transfection reagenta is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high e. These cells, an ad5 transfromed human embryonic kidney cell line containing a copy of the sv40 large t antigen, can be transfected very efficiently by the calciumphosphate method 75100%. Apr 24, 2012 be very gentle when feeding 293t cells. Higher cell densities may cause lower nucleofection efficiencies. Twentyfour hours prior to transfection, inoculate 1 to 2 x106 cells10 cm plate or, inoculate 3x106 cellsplate 16hours prior to transfection. Remove the cells from the tissue culture flasks following steps, subculturing cells. Once the culture has been started and the cells are growing normally, you should prepare frozen aliquots to provide a renewable source of cells. Cell culture primary mouse microglia, mouse astrocyte, neuro2a mouse neuroblastoma, 293t, nih3t3 and hela cells were cultured in dmem cellgro supplemented with 10% fetal bovine serum fbs and penicillinstreptomycin invitrogen.
Cell culture guidelines the following is a general guideline for culturing of cell lines. Hek293 cell line origins, cytogenetics, and expression. Suspension cell culture methodology adaptation to suspension culture was carried out using multiple methods table 1. Introduction tzmbl, 293s gnti and 293t17 cell lines are integral reagents for many aspects of the neutralizing antibody assay. They will detach from tissue culture treated plastic very easily.
Hek 293t cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for. Hek 293t tissue culture protocols brigham young university. Pipet a calculated aliquot of cell suspension into the new, unused 75 cm2 cell culture flask or 60 ml petri dish. Hek293 cells human embryonal kidney cells hek293, atcc cat. The protocol can be scaled to produce different amounts of virus as needed. When 293t cells were transplanted into the cleared fat pads of 3 week old scidbeige mice, all 1010 glands developed tumors at 14 days.
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