The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell culture. Aspirate and add fresh culture medium every 23 days. Transfection protocol lipofectamine 3000 transfection reagent. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Mammalian cell tissue culture techniques protocol abcam. In vitro cellular models of human hepatic fatty acid. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008. The performance of the hep2ifa test is strongly influenced by several technical details, including cell culture conditions, cell fixation and permeabilization methods, choice and titration of. In vitro cytotoxicity mtt assay in vero, hepg2 and mcf 7. The adherent cells are washed in 1 x hbs, and the precipitate is added dropwise to the culture dish. Other factors causing variability include growth time of the hep 2 culture, ph of the assay reagents, and many others. The hep 2 cell, a native protein array with hundreds of antigens, provides an ideal substrate for the detection of ana 1. Methods in molecular biology methods and protocols, vol 1250.
Avp00432 preparation for 3d cell culture on alvetex. Cell culture protocols thermo fisher scientific sg. Aspirate and add fresh culture medium every 2 3 days. Im gonna start to work with hepg2 cells and i would to know what are best culture conditions for these cells. Exposure of hep2 cells to stress conditions influences. Hepg2 stable cell line generation and rnai applications. Cells were fixed, embedded in paraffin wax, sectioned 10 m and. The cell culture must have 90% viability and be 7580% confluent on the day of transfection. Adherence of aeromonas caviae to hep2 and caco2 cell monolayers was investigated with 24 clinical isolates. Hep2 atcc ccl23 homo sapiens hela contaminant carcinoma.
The hep2 cell line has been described to originate from tumours which were produced. Given the there are many formulations of dmem and different versions of serum, we would like to provide the catalog numbers of. Stop enzyme activity with cm containing 10% serum and collect the cells in 15 ml conical tubes. The following is a general guideline for culturing of cell lines. Product information of hep2 cells cls cell lines service. Formation of biofilm on tissue culture plastic, hep 2 cells, and chicken intestinal epithelium. The ability of salmonella strains to form biofilms on the surface of hep 2 cells was investigated by using a modification of the flowthrough continuous culture system described previously by parsek and greenberg. Adherence of aeromonas caviae to human cell lines hep2.
Proper culture techniques and procedures are an essential part of ensuring successful transfection. Growth phase, temperature, multiplicity of infection and length of incubation affected. Hep 2 is a cell line derived from hela they were are contamination in an originally hepatic cell line see atcc for details and so are a cervix carcinoma cell line, most useful, because they have a rather big nucleus. Thawing should be rapid, approximately 2 minutes or until ice crystals have melted. Atcc confirmed this cell line is positive for the presence of human. Hep g2 or hepg2 is a human liver cancer cell line hep g2 is an immortal cell line which was derived from the liver tissue of a 15yearold african american adolescent boy with a welldifferentiated. Example protocol for the culture of the hepg2 cell line on alvetex 22 mm disc in 6well insert format, ams.
Detection of autoantibodies by indirect immunofluorescence. Formation of biofilm on tissue culture plastic, hep2 cells, and chicken intestinal epithelium. Screening for igg antinuclear autoantibodies by hep2. The adherent cells are washed in 1 x hbs, and the precipitate is added dropwise. The term antinuclear antibody ana describes a variety of autoantibodies that react with constituents of cell nuclei including dna, proteins and ribonucleoproteins 1,2. Communicable disease management protocol hepatitis b may 2018 2 ordering practitioner, and forwarded to the mhsal public health surveillance unit 2049483044 secure fax. These are normal cells derived from animal or human cells. So far ive had no problems whatsoever growing hepg2s in dmem, 10%fbs, penstrep, lglu. Pdf cell lines represent an invaluable resource in modern science including basic and translational cancer research. The term antinuclear antibody ana describes a variety of autoantibodies that react with constituents of cell nuclei including dna, proteins and ribonucleoproteins 1, 2. Ana testing should preferably use human epithelial2 hep2 cell line or rodent tissue as substrate. Hep g2 or hepg2 is a human liver cancer cell line hep g2 is an immortal cell line which was derived from the liver tissue of a 15yearold african american adolescent boy with a welldifferentiated hepatocellular carcinoma.
Take out required amount of cell suspension from the flask using pipette and place into new flask. Hepg2 liver hepatocellular carcinoma cell culture and. Add required amount of prewarmed cell culture media to fresh flask. Only 33% test their cell lines for cell identity 19692004 publications 10x, total 2. Volumes used in this protocol are for 75 cm 2 flask. Hep2 is a cell line derived from hela they were are contamination in an originally hepatic cell line see atcc for details and so are a cervix carcinoma cell line, most useful, because they have a rather big.
Antinuclear antibody screening using hep2 cells ncbi. Kathiresan2 1department of zoology, annamalai university, annamalai nagar, chidambaram608002, tamilnadu, india. Brightfield micrographs showing the structure of hepg2 cells cultured for 7 days on 22 mm diameter alvetex discs presented in the 6well plate format. Coculture of human liver macrophages and cholangiocytes. Phase contrast micrographs of hepg2 cells grown in. Chlorophyllin protects hep2 cells from nuclear fragmentation. Transfecting plasmid dna into hepg2 cells using lipofectamine 3000. After the cells of interest have been isolated from living tissue, they can. The effect of lamino acid oxidase on hep2 squamous cell. Applic on n hepg2 labeling and cell growth protocol for.
Sources of variability include preparation of slides, conjugate specificity, and efficiency of the fluorescent microscope and experience of the reader. All further steps are described in the subculture section. Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. Practice guidelines are systematically developed statements to assist practitioners and patients in making decisions about appropriate health care for specific clinical circumstances. Volumes used in this protocol are for 75 cm2 flask. Understanding and managing cell culture contamination. Brightfield micrographs showing the structure of hepg2 cells cultured for 7. Add appropriate aliquots of the cell suspension to new culture flasks and incubate cultures at 37. Culture and functional characterization of human hepatoma. Original article rljrgd4, an rgd toxin protein from.
Screening by hep 2 cells is a critical first step in the diagnosis of patients with sard. Hepg2 cell culturing protocol hepg2 complete medium. Note that cell culture conditions vary for each cell type. The 3 technologists noted differences owing to the percentage of mitotic cells, the cell size, the use or overuse of evans blue counterstain, the intensity of fluorescence, and the use of hep 2 cells transfected with 60kda ssa. Transfection protocol lipofectamine 3000 transfection. Hep g2 cells are passaged 24 hr prior to transfection to give a cell density of 5060% confluence at the time of transfection. Yost1,3 1 department of chemistry, university of florida, gainesville, fl. Material and methods virus and cell culture poliovirus type 1 obtained from the american type and culture collection atcc, number vr58, was grown in hep 2 cell cultures human larynx carcinoma, atcc. Applic on n hepg2 labeling and cell growth protocol for iroa. Eagles minimum essential medium emem supplemented with 10% fbs. Dec 12, 2018 the performance of the hep 2 ifa test is strongly influenced by several technical details, including cell culture conditions, cell fixation and permeabilization methods, choice and titration of fluorochromeconjugated secondary antibody, use and choice of blocking solutions, washing buffers, and antifading mounting medium. Adherence of aeromonas caviae to hep 2 and caco 2 cell monolayers was investigated with 24 clinical isolates. Transfection protocol transfection components component cat.
General details of cell culturing and subculturing. Hepg2 liver hepatocellular cells description, culturing medium conditions, transfection efficiency and protocols, and optimized transfection kits. May 25, 2009 immunofluorescence of the persistently infected hep 2 cell culture with monoclonal antibodies directed against any of four hrsv proteins g, f, n and p revealed significant heterogeneity in the expression level of viral antigens among individual cells, compared with a lytically infected culture fig. The human epitheliallike tumor line hep2 ccl23 was from the american type culture collection manassas, va. Sub culture volumes used in this protocol are for 75 cm2 andor 525 cm2 flask. In vitro cytotoxicity mtt assay in vero, hepg2 and mcf 7 cell lines study of marine yeast p. Propagation of hepg2 2 5 immediately remove cells and pellet at 500 xg for 5 minutes 4oc. Isolation and culture of liverderived macrophages and cholangiocytes for ldm isolation. 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.
Antinuclear antibody screening using hep2 cells protocol. Hep2 cells that survived a lytic infection with human respiratory syncytial virus hrsv were grown to obtain a persistently infected culture that produced relatively high amounts of virus 10. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Desired test nanomaterial concentration is determined from hep g2 hepatocyte cytotoxicity assay ncl method. For this purpose, the sample must be encapsulated in glissons capsula on all areas except, obviously, the edge left by the surgeon. A special cell line, hep2, qualified during the last years as substrate for. They are used for the primary isolation of viruses and production of vaccine. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. Lipofectamine 3000 transfection reagent hepg2 liver.
Hepg2 cells were routinely maintained in t75 flasks. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression. The ability of salmonella strains to form biofilms on the surface of hep2 cells was investigated by using a. Minimum leakage if any must occur during perfusion. So far ive had no problems whatsoever growing hepg2s in dmem. Monkey kidney cell culture, human amnion cell culture. Wst1 cell proliferation and cytotoxicity assay to determine the effects of rljrgd4 on hep2 cell proliferation, wst1 cell. Stop enzyme activity with cm containing 10% serum and collect the. Subculturing, also referred to as passaging, is the removal of medium and transfer of cells from a culture into fresh growth medium, in order to propagate the cells. So, if you need liver cells, hep 2 are a bad choice. Cell culture basics handbook thermo fisher scientific. Material and methods virus and cell culture poliovirus type 1 obtained from the american type.
Kathiresan2 1department of zoology, annamalai university, annamalai nagar. They are able to grow only for limited time and cannot be maintained in serial culture. Phase contrast micrographs of hepg2 cells grown in conventional 2d culture plates. Receipt of frozen cells and starting cell culture 1 immediately place frozen cells in liquid nitrogen freezer storage until ready to culture. Provided that iif is the first line screening tool for sard, understanding the steps to correctly perform this technique is critical. For this purpose, the sample must be encapsulated in glissons capsula on all areas except, obviously, the. Guidelines are provided for propogation of mammalian tissue culture cells.
Hepg2 cell optimum transfection condition optimum condition for transfection for 24well plate table 1 optimum condition for tranfection to hepg2 cells cell density serumfree medium 30 l dna 1. Dmem and rpmi1640 are also alternatives that work well. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent. H e p g 2 labe l ing and cell growth col hepg2 labeling and cell growth protocol for iroa phenotypic metabolic profiling candice z. Cell counting and seeding all cell manipulation steps in the culture hood. I only recently started to work in a hepatology department but hepg2 cell culture and transfection is fairly standard here. Cultures of hep2 cells persistently infected by human. Transfecting plasmid dna into hepg2 cells using lipofectamine.
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