Cell density was determined by an automatic cell counter coulter counter z2, beckman coulter, inc. Propidium iodide, indicating amount of dna stained per cell. Add cells to culture flask and incubate at 37c, 5% co2. Generation of lymphoblastoid cell lines lcls springerlink. This system integrates both the metabolism and the transport capacities of caco2 cells. Initiation and care of lymphoblast cell lines from human. Viable cell count was taken using trypan blue dye exclusion method at. The karyotype is pseudodiploid human male cell line. Split cells when the culture reaches a density of 1 x 106 cells per ml. Since lcl are latently infected with ebv, they provide a model system to investigate ebv latency and virusdriven b cell proliferation and tumorigenesis 1. An alternative method to establish lcls has been reported, which involves adding ebvcontaining culture supernatant on white blood cells. If they ever grow to more than 1,000,000 cellsml, they start slowing down.
Fundamental techniques in cell culture sigmaaldrich. Utility of lymphoblastoid cell lines for genetic and functional studies. Perform all steps, except centrifugation in a sterile tc hood using aseptic techniques. Infection of b cells with epsteinbarr virus ebv leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines lcl in vitro. Frozen cells will be received in 1 ml aliquots on dry ice. Cell culture basics handbook thermo fisher scientific.
Enhancer divergence and cisregulatory evolution in. Lymphoblastoid cell lines obtained from coriell biorepositories were. Cell culture human lymphoblast cell lines tk6, nh32, and. Lymphoblastoid cell lines lcls transformed by epsteinbarr virus ebv serve as an unlimited resource of human genomic dna. Efficient and reliable establishment of lymphoblastoid cell lines by. A detailed protocol for routine use is given for the establishment of lymphoblastoid lines from peripheral blood using espteinbarr virus and the immunosuppressivum cyclosporin a. Zhou and others published cell culture, bioreactors, commercial production. A routine method for the establishment of permanent. Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. Lymphoblastoid cell lines obtained from the coriell cell repositories were established by epsteinbarr virus.
If they are diluted to less than 200,000, they can start dying off. Steps involved in establishment of lymphoblastoid cell line with epstein barr virus. Extreme clonality in lymphoblastoid cell lines with implications for. Grow cells according to specific cell culturing conditions. Hyperglycemia, lymphoblastoid cell, gene mutations 1.
Nowadays, animal cell culture becomes a reasonable alternative for animal experiments in the process of drug discovery and development. How to count and calculate the number of cells from a stock flask or culture dish counting cells in a countess ii. Should the nutrients in the medium become exhausted too quickly, the adherent cells. On day 1, thaw b958 cells and culture in 6 ml complete rpmi 1640 medium. Care of lymphoblast cell lines from human peripheral blood. After one month of ebv infection, lymphoblastoid cell clusters are visible. If they ever grow to more than 1,000,000 cells ml, they start slowing down. Efficient and reliable establishment of lymphoblastoid cell lines by epsteinbarr virus transformation from a limited amount of peripheral blood. However, it is recommended that if you are going to store the cells for later use, grow up fresh stocks and freeze multiple ampoules. What are the basic culture conditions for lymphoblasts. Lymphoblastoid cell an overview sciencedirect topics. For lymphocytes, transformation by ebv is a method of choice as it imparts. The aim of this protocol is to describe the shipment of biological material, dna, lymphoblastoid cell lines at room temperature and in dry ice cell line vial. Because cloning of lcl cells by conventional culture is usually difficult, they used.
Use flask appropriate for the amount of cells being cultured. General protocol for the immortalization of human blymphocytes. Animal cell culture protocol aseptic technique and good cell culture practice to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. We started cell culture by thawing a frozen cell stock in 50ml medium with ts75 culture flask and expansively cultured the cells for experimental use. Lymphoblastoid cell lines obtained from coriell biorepositories were established by. Lymphoblast cell culture protocols are listed within our website, regarding from shipment of frozen cells and live cultures. This protocol describes the materials and methods we use to culture mouse mast cells. The modal chromosome number is 46, occurring in 74% with polyploidy at 5. Encyclopedia of industrial biotechnology find, read and cite all the research you need on.
If the lymphoblast cells have excessive red blood cell. For these types of cell lines subculture by dilution is relatively easy. Jurkat atcc number tib152 cell culture and formaldehyde. A simple red blood cell lysis method for the establishment of b. Xpdl lymphoblastoid cells following h2o2 treatment. Cultures should be seeded at a concentration of no less than 200,000 viable cells ml. General proceduresmaintenance of cell line protocols.
Genotoxicity assay on a human coculture cell system. Culture of blymphoblastoid cell lines view product listing ask a question. Effros, in advances in cell aging and gerontology, 2002. Chromosome preparation from cultured cells protocol. The method described in this paper generates lcl from donor peripheral blood with rapid immortalization.
Fetal calf serum lymphoblastoid cell line rpmi complete medium virus. 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 clone e61 is a human t lymphoblastoid cell line derived from an acute t cell leukemia. As a first step in the assessment of h 2o 2 as an adjuvant treatment for csi, we studied the ability of h 2o 2 to kill chondrosarcoma cell lines in vitro. Protocol for caco2 cell culture protocol online this protocol describes about how caco2 cells should be maintained into the labs and common cares which should be taken during culture process. A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture counting cells in a hemocytometer. The property of lcls to be able to grow in continuous culture together by maintaining a close similarity to the starting parent lymphocytes has been very well exploited in various studies leading to large body of literature. Pdf establishment of epsteinbarr virus growthtransformed. Expression of transfected dna and selection of transfected cells, somatic cell and molecular genetics, vol. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. This protocol assumes the investigator is using proper sterile tissue culture techniques. E 1983 chromosome site for epsteinbarr virus dna in a burkitt tumor cell line and. An important point when you start a new culture, put your t25 flask in a vertical position, so cells will be in closer contact than in horizontal position.
Lcl have been used to present antigens in a variety of. These aggregates can be dissociated by gently agitating the culture or by gentle. When the cells have reached logarithmic phase approximately 2 days after cells are split, add 10. Chromosome harvesting of lymphoblastoid cell lines. We have developed a longterm t cell culture system in which human t cells are followed from the point of primary stimulation to the endpoint of replicative senescence. Transfection of lymphoblastoid cells using dnaloaded. Permanent lymphoblastoid cell lines are of great practical value in human clinical and experimental genetics.
Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. Epstein barr virus transformation of peripheral blood mononuclear cells using. A routine method for the establishment of permanent growing. Keep cells frozen until thawed for cell culture or place in liquid nitrogen storage if not starting cultures right away. Over the last three decades, ecacc has expanded and diversified to become one of the. In addition, the biologic basis of this transformation system is briefly summarized. Lymphoblastoid cell lines grow in suspension culture with cells clumped in loose. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or medium that supplies. This protocol gives you the preferred medium and culture conditions that will ensure success.
The european collection of authenticated cell cultures ecacc the european collection of authenticated cell cultures ecacc was established in 1985 as a cell culture collection to service the research community and provide an international depository authority recognized patent depository for europe. Immune cell protocols, research techniques astarte biologics. Establishment of epsteinbarr virus growthtransformed. Culturing cryopreserved lymphoblastoid cell lines 1. Lymphoblast culture faq form 214 rev e092415 page 1 of 2 1. Permanent lymphoblastoid cell lines are of great practical value in human. Our blymphoblastoid cell lines blcl are easy to grow, but instructions are still helpful. Culture of blymphoblastoid cell lines astarte biologics. Dna damage was investigated using the comet and micronucleus assays on human intestinal cells caco2 cells and on tk6 cells, which represent the blood compartment. Lymphoblastoid cell lines obtained from the coriell cell repositories were established by epsteinbarr virus transformation of peripheral blood mononuclear cells using phytohemagluttinin as a mitogen.
Efficient and reliable establishment of lymphoblastoid. Cells grow in suspension and may form large cell clusters. Overall, an aspect of pharmaceutical research which promisingly employs cell culture models is the study of in vitro drug transportabsorption and metabolism. Immunology, issue 119, ebv, red blood cell lysis method. Revised and approved 2016feb18 pc harbor ficoll ficollpaque plus. We used our standard protocol to carry out mutagenesis study 18. Pdf hydrogen peroxide induced genomic instability in.
Cell culture protocols thermo fisher scientific kr. Introduction the prevalence of diabetes mellitus is increasing. All cells lines are free of bacterial, fungal or mycoplasma contamination. Lymphoblast cell culture protocols the progeria research. The culture system involves the use of antigenpresenting cells apc rather than antibodies to cd3 and cd28, because of the difference in. Drexler, in the leukemialymphoma cell line factsbook, 2001. How to count and calculate the number of cells using an automated cell counter. Care of lymphoblast cell lines from human peripheral blood note.
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