Transfection of jurkat cells use this procedure to transfect plasmid dna into jurkat cells in a 24well format for other formats, see scaling up or down transfections, below. Harvestingcells formaldehyde%crosslinked%cells%for%chromatin%immunoprecipitation% 1. Different derivatives of the jurkat cell line that have been mutated to lack certain genes can now be obtained from cell culture banks. Jurkat cells can produce interleukin 2, and are used in research involving the susceptibility of cancers to drugs and radiation. Immunoisolation of tcr signaling complexes from jurkat t.
Stable neomycin neoresistant single cell originated clones were isolated and expanded by selection in medium containing 1 mgml g418 for 14 days 1. Cultures can be maintained by the addition of fresh medium or replacement. Invitrogen geneblazer jurkat control kit assay protocol o12776. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. In addition to tabalugas comments, i would like to add that, selecting the cells that are living via these methods is a sort of selection pressure on the surviving cells, which means that the jurkat cells you now have are no longer jurkats so unless these cells are particularly precious e. Remove and discard 10 ml of old growth medium from above the cell pellet. We have developed a method to immunoisolate activated t cell receptors tcrs and associated signaling molecules in plasma membrane subdomains from jurkat t leukemic cells. Supplemental experimental procedures cell culture jurkat cells. The initial propagation of cells should be used to generate stocks to be frozen and stored for future use.
Essential role of the adaptor protein nck1 in jurkat t. How to count and calculate the number of cells using an automated cell counter. Jurkat t cells 210 4 cells in 90 ul medium were seeded in 96well clear plates for overnight. Sub culture and maintenance 1 take cell counts with a hemocytometer every 48 hours to maintain the culture at a cell density between 1x105 and 1x106 cellsml maximal density is at 3x106 cellsml.
A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture counting cells in a hemocytometer. Once they are established, grow the cells in techne stirrer bottlesthis allow you to grow 100s of millions of cells for your experiments. Nfatbla jurkat cellbased assay catalog number k1710 doc. Jurkat cells were treated with pha for 20 hours and secreted il2 was analyzed alone or in the presence of jurkat cells. Read the safety data sheets sdss and follow the handli. The formation of multimolecular assemblies of signaling molecules at the plasma membrane is key to triggering signaling cascades following activation of transmembrane receptors at the plasma membrane. Jurkat cells were maintained in rpmi1640 medium invitrogen supplemented with 10% fetal bovine serum, containing penicillin 100 uml. Chimeric antigen receptor car development involves extensive empirical characterization of antigenbinding domain abdcar constructs for clinical suitability. Subculturing jurkat cell line protocol for use with c2009 and c2010 transfer growing culture from t75 flask to a sterile 50 ml conical tube. Hi can anyone please give me a detailed proceduregeneral protocol for cell culture of suspension cells non adherent cells.
Successful crispr genome editing in hardtotransfect. Stable neomycin neoresistant singlecelloriginated clones were isolated and expanded by selection in medium containing 1 mgml g418 for 14 days 1. But at first when they are recovering from cryopreservation, go for 1. Here, we present a costefficient and rapid method for evaluating cars in human jurkat t cells.
The assay was read on an ique screener, following the no wash protocol. Precipitate 5 pmol around 10 g plasmid dna in 100 l. Successful crispr genome editing in hardtotransfect cells. The modal chromosome number is 46, occurring in 74% with polyploidy at 5.
To define the impact of mpa on the metabolism of proliferating t cells, we incubated jurkat t cells a validated model for studying the. All amounts and volumes are given on a per well basis. T lymphocyte, acute t cell leukemia growth properties. Discard supernatant, resuspend cells in 20 ml prewarmed complete cell culture medium and transfer cells into one t75 cell culture flask. Tflasks 25 ml culture volume to a cell density of approxi. Mediashouldbechanged1daypriortoharvestingtoinsurecellsare%. Jurkat atcc number tib152 cell culture and formaldehyde cross linking. Jurkat single cell clonal expansion biology stack exchange. Adcc induction below is a protocol for endpoint readings using a. Which supplement is needed for growing of jurkat cells.
Jurkat, clone e61 atcc tib152 homo sapiens peripheral b. Splitting can be performed by centrifuging cells at 500g for 5 minutes, decanting growth medium and rinsing in sterile 1x pbs. Hence im wondering if anyone has any protocols they could suggest such as contents of the media e. Jurkat clone e61 is a human t lymphoblastoid cell line derived. To ensure reproducible results, homogenize the cell suspensions. Receipt of frozen cells and starting cell cultures.
Dorschner ordering information jurkat can be ordered from atcc as a frozen ampoule. Gibco media, sera, supplements, cells, and cell culture reagents are designed to deliver reproducibility and performance for results you count on. Simultaneous analysis of secreted il2 and jurkat cells. An il2 producing cell line, derived by incubating the cells at 41c for 48 hours followed by a limiting dilution cloning over macrophages. Maintain the cell density of the culture as suggested under the subculture procedure. Cd25 expression on the surface of jurkat cells springerlink. Cell growth protocol for jurkat cell line jurkat ucsc genome. Antibcma car nfat luciferase reporter jurkat cell line. Flow cytometric detection of pakt and foxp3 in jurkat t.
Cell culture guidelines the following is a general guideline for culturing of cell lines. Cell culture protocols thermo fisher scientific in. The jurkat cell is an immortalized line of human acute. This reference provides a recommended procedure to transfect plasmid dna into human jurkat human tcell leukemia cells atcc cat. Norma neff and tim reddy jurkat atcc number tib152 cell culture and formaldehyde crosslinking jurkat clone e61 is a human t lymphoblastoid cell line derived from an acute t cell leukemia. Jurkat cells are an immortalized line of human t lymphocyte cells that are used to study acute t cell leukemia, t cell signaling, and the expression of various chemokine receptors susceptible to viral entry, particularly hiv. I am fully aware that there might not be a single right answer to this and the answer requires a degree of experienceopinion on the working method. Using a modular car plasmid, a highly efficient abd cloning strategy, plasmid electroporation, shortterm coculture, and flowcytometric. Flow cytometric detection of pakt and foxp3 in jurkat t cells. Resuspend the cells carefully in 10ml fresh cell culture medium and transfer them into one t25 cell culture flask. Transfer oneday old growing culture into a sterile 50 ml conical tube. Jurkat cells revealed high sensitivity to arac cultured on tcps. Determination of the membrane transport properties of jurkat cells. Split cells, if necessary, to obtain optimal confluency for electroporation.
This cell line grows in suspension and should be maintained at a density between 1x105. As is the case today, tcell biologists in the early 1980s focused their research efforts mainly on cells of human or mouse origin. The karyotype is pseudodiploid human male cell line. The jurkat cell line originally called jm was established in the late 1970s from the peripheral blood of a 14yearold boy with t cell leukemia.
Jurkat t cells and development of the tcell receptor. Change the cell culture media on the cells 1 day before electroporation. Il2 secretion and cell counts from costimulated jurkat cells. To obtain the desired infection rate low and high moi. Invitrogen k1186 cellsensor isrebla jurkat cellbased assay protocol k1186. This protocol assumes that the cells are being cultured in t150 flasks equipment equipment. Remove the frozen vial of cells from liquid nitrogen and quickly thaw them by swirling in a 37. Jurkat cell tissue and cell culture protocol online. A cell line derived from human tcell leukemia and used to determine the mechanism of differential susceptibility to anti. Bcl2 jurkat atcc crl2899 homo sapiens peripheral blood a. The jurkat cell line was established from the peripheral blood of a 14 year old boy by schneider et al. Regardless of whether you are performing cell culture in a research lab or production facility, need a special or defined formulation, or are growing cell lines, primary cells, or stem cells. Sub culture and maintenance 1 maintain culture at a cell density between 1x105 and 3x106 cellsml.
The jurkat cell line has most often been used as a prototypical t cell line to study multiple events in t cell biology, including a t cell signaling and. Essential role of the adaptor protein nck1 in jurkat t cell. Il2 production assay in jurkat tcells stimulated with. Do not use freshly thawed cells for electroporation. For transient transfection of jurkat e61 cells, we recommend the lonza nucleofector device and the cell line nucleofector kit v using cell typespecific protocol. Jurkat clone e61 is a human t lymphoblastoid cell line derived from an acute t cell leukemia. This is a clone of the jurkat fhcrc cell line, a derivative of the jurkat cell line. Centrifuge jurkatlucia nfat cd16 cells at 800 rpm rcf 150 g for 5 minutes. Remove supernatant and resuspend jurkatdual cells at 2 x 106 cellsml in fresh, prewarmed test. Subculture and maintenance 1 take cell counts with a hemocytometer every 48 hours to maintain the culture at a cell density between 1x105 and 1x106 cellsml maximal density is at 3x106 cellsml. Start the cells in tissue culture flasks, for example a t75.
This is a clone of the jurkatfhcrc cell line, a derivative of the jurkat cell line. This protocol is for transduction of suspension cells jurkat t cells, pbmc, pbl, b cells etc. Thawing cells use the following protocol to thaw nf. The cells produce large amounts of il2 after stimulation with phorbol esters and either lectins or monoclonal antibodies. The day of transfection, count the cells to determine culture density. Sub culture and maintenance 1 take cell counts with a hemocytometer every 48 hours to maintain the culture at a cell density between 1x105 and 1x106 cells ml maximal density is at 3x106 cells ml. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. Jurkat atcc number tib152 cell culture and formaldehyde. Each of these steps, and many other intermediate factors in this pathway, has been dissected using mutant subclones of the jurkat t cell line, which has been. Spin cells down for 5 min at 200 x g at room temperature rt. Below is a protocol for endpoint readings using a luminometer with an injector, this protocol can be adapted for use with kinetic measurements or a luminometer with a manual setup.
Monty montano, in translational biology in medicine, 2014. American type culture collection, rockville, md, usa were grown in the rpmi1640 medium g ibco, gaithersburg, md, usa supplemented with 10% heatinactivated fetal bovine serum fbs g ibco, 100 iuml penicillinstreptomycin jrh biosciences, victoria, australia, and 2 m m lglutamine jrh biosciences at 37c in the humidified atmosphere with 5%. Centrifuge cells at 800 rpm rcf 150 g for 5 minutes. Jurkat transfection by electroporation odified, j exp med. The jurkat cell line has most often been used as a prototypical t cell line to study multiple events in t cell biology, including a t cell signaling and b molecular events in the hiv. Cck8 reagent was used to test cell cytotoxicity of jurkat cells cultured on tcps, ups and 80 gml ctcps.
Using a modular car plasmid, a highly efficient abd cloning strategy, plasmid electroporation, shortterm co culture, and flowcytometric detection of cd69, this assay referred to as carj evaluates sensitivity and specificity for abds. Culture should be approximately 90% confluent prior to preparation of frozen stock in order to insure the highest number of viable cells from reviving a frozen culture. The jurkat cell line has been cloned into numerous sublines. For these experiments, we used clone e61 jurkat cells atcc tib152.
How to count and calculate the number of cells from a stock flask or culture dish counting cells in a countess ii. Jurkat cells can be fixed as follows for the detection of pakt. Jurkat, e6, jurkat e6, e61 human leukaemic t cell lymphoblast derived from jurkat fhcrc. Mpa decreases cell viability and promotes apoptosis.
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