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. So, if you need liver cells, hep 2 are a bad choice. 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. Hepg2 cell culturing protocol hepg2 complete medium. Provided that iif is the first line screening tool for sard, understanding the steps to correctly perform this technique is critical. After the cells of interest have been isolated from living tissue, they can. Stop enzyme activity with cm containing 10% serum and collect the cells in 15 ml conical tubes. Material and methods virus and cell culture poliovirus type 1 obtained from the american type.
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. 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. Guidelines are provided for propogation of mammalian tissue culture cells. In vitro cytotoxicity mtt assay in vero, hepg2 and mcf 7 cell lines study of marine yeast p. Original article rljrgd4, an rgd toxin protein from. Kathiresan2 1department of zoology, annamalai university, annamalai nagar. Screening for igg antinuclear autoantibodies by hep2. Isolation and culture of liverderived macrophages and cholangiocytes for ldm isolation. The term antinuclear antibody ana describes a variety of autoantibodies that react with constituents of cell nuclei including dna, proteins and ribonucleoproteins 1, 2. In vitro cellular models of human hepatic fatty acid.
Formation of biofilm on tissue culture plastic, hep 2 cells, and chicken intestinal epithelium. Eagles minimum essential medium emem supplemented with 10% fbs. Culture and functional characterization of human hepatoma. All further steps are described in the subculture section. Cell culture basics handbook thermo fisher scientific. Hepg2 liver hepatocellular cells description, culturing medium conditions, transfection efficiency and protocols, and optimized transfection kits. Add required amount of prewarmed cell culture media to fresh flask. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression. Hep2 atcc ccl23 homo sapiens hela contaminant carcinoma. Detection of autoantibodies by indirect immunofluorescence. 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. In vitro cytotoxicity mtt assay in vero, hepg2 and mcf 7.
Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent. Applic on n hepg2 labeling and cell growth protocol for. Pdf cell lines represent an invaluable resource in modern science including basic and translational cancer research. The cell culture must have 90% viability and be 7580% confluent on the day of transfection. The adherent cells are washed in 1 x hbs, and the precipitate is added dropwise to the culture dish.
Hepg2 stable cell line generation and rnai applications. So far ive had no problems whatsoever growing hepg2s in dmem. Adherence of aeromonas caviae to hep2 and caco2 cell monolayers was investigated with 24 clinical isolates. Antinuclear antibody screening using hep2 cells protocol. 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. Brightfield micrographs showing the structure of hepg2 cells cultured for 7 days on 22 mm diameter alvetex discs presented in the 6well plate format. 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. Minimum leakage if any must occur during perfusion.
Sources of variability include preparation of slides, conjugate specificity, and efficiency of the fluorescent microscope and experience of the reader. Receipt of frozen cells and starting cell culture 1 immediately place frozen cells in liquid nitrogen freezer storage until ready to culture. The effect of lamino acid oxidase on hep2 squamous cell. Adherence of aeromonas caviae to hep 2 and caco 2 cell monolayers was investigated with 24 clinical isolates.
The hep 2 cell, a native protein array with hundreds of antigens, provides an ideal substrate for the detection of ana 1. Cell counting and seeding all cell manipulation steps in the culture hood. They are used for the primary isolation of viruses and production of vaccine. 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. Transfection protocol lipofectamine 3000 transfection. So far ive had no problems whatsoever growing hepg2s in dmem, 10%fbs, penstrep, lglu. Note that cell culture conditions vary for each cell type. For this purpose, the sample must be encapsulated in glissons capsula on all areas except, obviously, the.
Proper culture techniques and procedures are an essential part of ensuring successful transfection. Sub culture volumes used in this protocol are for 75 cm2 andor 525 cm2 flask. Propagation of hepg2 2 5 immediately remove cells and pellet at 500 xg for 5 minutes 4oc. Hepg2 liver hepatocellular carcinoma cell culture and. Volumes used in this protocol are for 75 cm2 flask. Hepg2 cells were routinely maintained in t75 flasks.
The hep2 cell line has been described to originate from tumours which were produced. 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. Wst1 cell proliferation and cytotoxicity assay to determine the effects of rljrgd4 on hep2 cell proliferation, wst1 cell. Hep g2 cells are passaged 24 hr prior to transfection to give a cell density of 5060% confluence at the time of transfection. Volumes used in this protocol are for 75 cm 2 flask.
Avp00432 preparation for 3d cell culture on alvetex. Phase contrast micrographs of hepg2 cells grown in. Brightfield micrographs showing the structure of hepg2 cells cultured for 7. Understanding and managing cell culture contamination.
Aspirate and add fresh culture medium every 23 days. Desired test nanomaterial concentration is determined from hep g2 hepatocyte cytotoxicity assay ncl method. Applic on n hepg2 labeling and cell growth protocol for iroa. Kathiresan2 1department of zoology, annamalai university, annamalai nagar, chidambaram608002, tamilnadu, india. Atcc confirmed this cell line is positive for the presence of human. 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. Phase contrast micrographs of hepg2 cells grown in conventional 2d culture plates. They are able to grow only for limited time and cannot be maintained in serial culture. Ana testing should preferably use human epithelial2 hep2 cell line or rodent tissue as substrate. Antinuclear antibody screening using hep2 cells ncbi.
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. The term antinuclear antibody ana describes a variety of autoantibodies that react with constituents of cell nuclei including dna, proteins and ribonucleoproteins 1,2. Example protocol for the culture of the hepg2 cell line on alvetex 22 mm disc in 6well insert format, ams. Formation of biofilm on tissue culture plastic, hep2 cells, and chicken intestinal epithelium. Exposure of hep2 cells to stress conditions influences. Add appropriate aliquots of the cell suspension to new culture flasks and incubate cultures at 37.
The ability of salmonella strains to form biofilms on the surface of hep2 cells was investigated by using a. Product information of hep2 cells cls cell lines service. 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. General details of cell culturing and subculturing. Transfecting plasmid dna into hepg2 cells using lipofectamine. Adherence of aeromonas caviae to human cell lines hep2. Cultures of hep2 cells persistently infected by human. Stop enzyme activity with cm containing 10% serum and collect the. Other factors causing variability include growth time of the hep 2 culture, ph of the assay reagents, and many others. 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. Dmem and rpmi1640 are also alternatives that work well. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008.
Take out required amount of cell suspension from the flask using pipette and place into new flask. Mammalian cell tissue culture techniques protocol abcam. These are normal cells derived from animal or human cells. Chlorophyllin protects hep2 cells from nuclear fragmentation.
All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. The human epitheliallike tumor line hep2 ccl23 was from the american type culture collection manassas, va. Cells were fixed, embedded in paraffin wax, sectioned 10 m and. Growth phase, temperature, multiplicity of infection and length of incubation affected. 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.
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. Transfecting plasmid dna into hepg2 cells using lipofectamine 3000. Given the there are many formulations of dmem and different versions of serum, we would like to provide the catalog numbers of. Coculture of human liver macrophages and cholangiocytes. Monkey kidney cell culture, human amnion cell culture. 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. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. The adherent cells are washed in 1 x hbs, and the precipitate is added dropwise. 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.
Communicable disease management protocol hepatitis b may 2018 2 ordering practitioner, and forwarded to the mhsal public health surveillance unit 2049483044 secure fax. Thawing should be rapid, approximately 2 minutes or until ice crystals have melted. Methods in molecular biology methods and protocols, vol 1250. 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. Only 33% test their cell lines for cell identity 19692004 publications 10x, total 2. Cell culture protocols thermo fisher scientific sg. Lipofectamine 3000 transfection reagent hepg2 liver.
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. Yost1,3 1 department of chemistry, university of florida, gainesville, fl. I only recently started to work in a hepatology department but hepg2 cell culture and transfection is fairly standard here. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Screening by hep 2 cells is a critical first step in the diagnosis of patients with sard. Transfection protocol transfection components component cat. The following is a general guideline for culturing of cell lines. Transfection protocol lipofectamine 3000 transfection reagent.
887 1249 98 88 303 1438 453 412 1494 912 711 370 387 349 1509 900 116 377 315 407 66 314 356 1464 683 908 969 550 1219 237 388 823 345 1052 724 327 344 1026 606 106 1419 381 1395 453