Jurkat clone e61 is a human t lymphoblastoid cell line derived from an acute t cell leukemia. 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. Adherent cells are anchoragedependent and propagate as a monolayer attached to the cell culture vessel. However, adherent cell processes are often limited by a lack of advances in cell culture systems.
Not for use in diagnostic or therapeutic procedures. Cells can be harvested on day 8, or step 10 can be repeated to harvest cells on day 9 or 10. Apr 01, 2014 automated and online characterization of adherent cell culture growth in a microfabricated bioreactor article pdf available in journal of the association for laboratory automation 195 april. This protocol assumes that the cells are being cultured in t150 flasks equipment equipment. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Cell culture is the growth of cells from an animal or plant in.
Basic cell culture protocols basic cell culture protocols. A widely used protocol involves the culture of murine bone marrow bm cells with granulocyte. First, cell culture acquisition is performed with a lensfree video microscope, and then the data is analyzed following a fourstep process. This protocol has been adapted for a 12well plate system. Cells can be treated with drugs or subject to other manipulations, such as sirnamediated gene knockdown, as they would in regular cell culture protocols, prior to fixation and microscopy. Formalin and methanol are commonly used as fixing agents. The thawing of mef follows steps 15 outlined for thawing the p815 cell. The viability of cell banks is dependent on the cryopreservation procedure employed when making them and on the proper storage conditions. Measuring survival of adherent cells with the colony. The thermo scientific nunc cell factory system is a large multilayer unit designed for the culture of adherent cells. Fix the cells with 24% paraformaldehyde pfa for 1020 min at rt or with cold methanol, acetone 110 min at 20c. Suspension cell lines often have a recommended subculture seeding density. Automated and online characterization of adherent cell.
Cell culture protocol for passaging and splitting semiadherent cell lines using trypsin edta. Data analysis is a fourstep process based on multiwavelength holographic reconstruction, cell tracking, cell segmentation and cell division algorithms. Mammalian cell tissue culture techniques protocol abcam. Here we provide recommendations for characterizing a model assay system and some of the factors to consider when choosing cell based assays for manual or automated systems. Discard the cell culture medium by inverting the slide and gently tapping it on a paper towel to remove the remaining medium. The modal chromosome number is 46, occurring in 74% with polyploidy at 5. Culturetrax software enables you to quickly and easily plan and fully document your cell culture and analytical work. Use aseptic technique to prevent microbial contamination. Cell culture basics, techniques and media essentially, cell culture involves the distribution of cells in an artificial environment in vitro which is composed of the necessary nutrients, ideal temperature, gases, ph and humidity to allow the cells to grow and proliferate.
Cell culture basics handbook thermo fisher scientific. Aspirate culture medium and rinse the cells with pbs twice. To adapt this protocol for your preferred system, volumes should be adjusted accordingly. At this point the cell lines should be subcultured or passaged in order to prevent the culture dying.
Confluency confluency is a marker for when to subculture your cells. Harvesting adherentdependent cells from thermo scientific. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. A widely used protocol involves the culture of murine bone marrow bm cells with granulocytemacrophage colonystimulating factor gmcsf to generatebmderiveddcsbmdcs. Including cell culture basics, equipment, protocols and troubleshooting. Unless they undergo an immortalization procedure, primary cells have a limited lifespan and usually reach senescence after 1020 passages. Immobilization of nonadherent cells with celltak for.
Protocol for trypsinedta treatment for disruption of cell. For adherent cells that usually means reaching 8090% confluence there is still. The following protocol is suggested for the basic instruction of culturing adherent cell lines. The handbook is intended as a guide rather than an indepth text book of cell culture and you are encouraged to consult relevant specialised literature to obtain more detailed information. Note that the procedure for passaging insect cells differs from that for mammalian cells on several crucial steps. Immobilization of nonadherent cells with celltak for assay. Immunofluorescence protocol for adherent cells materials. May 21, 2018 the handbook and videos provide an introduction to cell culture, with a focus on maintaining cell health throughout the processes of culturing, freezing, thawing and passaging cells. To subculture the cells they need to be brought into suspension. Washing adherent cells in agilent seahorse xf24 cell culture. 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.
We describe the full protocol used to monitor and quantify a hela cell culture for 2. The following protocol describes a general procedure for subculturing adherent mammalian cells in culture. There are some instances when cell cultures may grow as semiadherent cells, e. A nontreated surface is also available for suspension or nonadherent cell culture and may also be used to study cellcell or cellprotein interactions in an in vitro system. This cell seeding protocol was developed by agilent seahorse bioscience scientists, and is applicable exclusively to cells cultured in the seahorse xf96 cell culture microplates or seahorse xfp cell culture miniplates coated with celltak, and is intended for analysis using a seahorse xfexf96 or xfp analyzer. Protocol for trypsinedta treatment for disruption of cell monolayers november 2018 i. Hek 293293t 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 episomal replication of plasmids containing the sv40 origin of replication notes hek cells are loosely adherent, and do not require digestion. Pdf automated and online characterization of adherent cell. Fix the cells with 4% formaldehyde diluted in 1x pbs prepare fresh for 10 min at room temperature fixation time can be increased to 20 min depending on the cell line. Adherent cells can be grown directly on chamber slides or coverslips. The design of a nunc cell factory system is similar to a cell culture flask, therefore, a standard cell harvesting protocol may be used. Detailed immune cell killing protocol for adherent target cells detailed demonstration protocol day 0 1 remove serumcontaining medium from skov3. Jurkat atcc number tib152 cell culture and formaldehyde. Measuring survival of adherent cells with the colonyforming.
Replace with new culture medium to the original volume. Detailed immune cell killing protocol for adherent target cells detailed demonstration protocol day 0 1 remove serumcontaining medium from skov3 target cell culture and gently rinse twice with. Detailed immune cell killing protocol for adherent target. Washing adherent cells in agilent seahorse xf24 cell. The handbook and videos provide an introduction to cell culture, with a focus on maintaining cell health throughout the processes of culturing, freezing, thawing and passaging cells. Bmdcsexpress cd11candmhcclassiimhciimoleculesandshare. Annexin v staining of adherent cells for flow cytometry. All cell culture procedures must be conducted in a biosafety cabinet. While naturally adherent cells such as fibroblasts and keratinocytes can be grown directly on plastic cell culture plates or glass coverslips and are resistant to repeated washing, cells cultured in suspension, such as lymphocytes, must be purposely adhered during the immunostaining procedure.
Trypsinization of adherent cells 1 observe adherent cells microscopically shake flask gently on the platform to assess adherence if too many cells are floating, it is an indication of low viability 2 discard medium into a waste beaker spray beaker with ethanol before introducing into hood wash x2 with pbs 3 ml each wash. Hek cells are loosely adherent, and do not require digestion by trypsinedta prior to being passaged. The morphology of the cells being cultured depends on. Protocol for adhesion and immunostaining of lymphocytes. Fundamental techniques in cell culture sigmaaldrich. Any and all media, supplements, and reagents must be sterilized by filtration through a 0. C2c12 cell culture, differentiation treatment, and crosslinking protocol. In order to explain this methodology based on lensfree video microscopy, we describe the protocol to monitor and quantify a hela cell culture for 2. Therefore to maintain this heterogeneity both the attached cells and the cells in suspension must be subcultured. An alizarin redbased assay of mineralization by adherent. Altogether, these results began to suggest that two types of loosely adherent.
Lensfree video microscopy for the dynamic and quantitative. Culturetrax technology that moves your stem cell science. It offers a repository for detailed, current protocols and recipes, structured to drive accurate execution in the lab and rapid, comprehensive recording of results. The cell line c2c12 is an immortal line of mouse skeletal myoblasts originally derived from satellite cells from the thigh muscle of a two month old female c3h mouse donor 70h after a. In cell culture the technique used to separate cells from tissue, there are two basic methods for culturing cells. For the extraction and expansion of ls1 cells, a 2g tissue biopsy from an osteoblastic tumor was recovered.
Adherent cell lines are widely used across all fields of biology, including drug discovery, toxicity studies, and regenerative medicine. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. The karyotype is pseudodiploid human male cell line. This cell seeding protocol was developed by agilent seahorse bioscience scientists, and is applicable exclusively to cells cultured in the seahorse xf96 cell culture microplates or seahorse xfp cell culture miniplates coated with cell tak, and is intended for analysis using a seahorse xfexf96 or xfp analyzer.
With minor modifications a standard protocol can be optimized for the unique. Please use one of the following formats to cite this article in your essay, paper or report. B958 marmoset where a proportion of cells do not attach to the tissue culture flask and remain in suspension. General details of cell culturing and subculturing. For choosing and preparing the appropriate assay medium, please see. Detailed immune cell killing protocol for adherent target cells.
Always check the guidelines for the cell line in use. Cell culture protocol for passaging and splitting semi adherent cell lines using trypsin edta. Depending on their origin, animal cells grow either as an adherent monolayer or in suspension. This section provides useful hints for culturing animal cells i. A confluent monolayer is an adherent cell culture dish, plate or flask in which the cells have formed a single layer over the entire surface area available for growth.
Cell lines are routinely frozen to make and keep referenceparental cell lines, newly produced transgenic cell lines, keep stocks of primary and immortalized cells, and for shipping purposes. For these types of cell lines subculture by dilution is relatively easy. Subculturing adherent cells thermo fisher scientific in. Falcon nontreated polystyrene hydrophobic surface with low to moderate binding properties. The following is a general guideline for culturing of cell lines. Atcc tracks the pdl and passage number for many adherent cell lines when the. Responsibility it is the responsibility of all trained laboratory personnel to perform and adhere to all aspects of the procedure. It covers different types of animal cell cultures, considerations for cell culture, and cell culture protocols.
Nonadherent and loosely adherent cells in the culture supernatant can be. Annexin v staining of adherent cells for flow cytometry for research use only. Cell culture guidelines the following is a general guideline for culturing of cell lines. Introduction adherent cell lines must be disrupted from cell culture flasks in order for them to be added to the neutralization assay or used for transfection for the production of pseudoviruses. The general protocol for these homogeneous assays is add, mix and measure. Oct 15, 2019 please use one of the following formats to cite this article in your essay, paper or report. General protocol for recovering or freezing primary cells. Culture guidelines of adherent cells using trypsin. This attachment is essential for proliferation many adherent cell cultures will cease proliferating once they become confluent i. Establishing an in vitro model system the species of origin and cell types used in cytotoxicity. Cell growth protocol and differentiation treatment for the. Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients.
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