All further steps are described in the subculture section. Do you have a protocol for transfection of macrophage cell. To give you confidence in the health of your cells every step of the way, weve highlighted the technologies and products within cell biology that are critical to maintaining optimal cell health. Cell line authentication str profiling aids in the detection of misidentified, crosscontaminated, or genetically drifted cells, which invalidate research results.
Can anyone explain why sometimes macrophages from cell line j774. Maintaining healthy cells is the key to experimental success and reproducible research results. Hepg2 is an immortalized cell line consisting of human liver carcinoma cells, derived from the liver tissue of a 15yearold caucasian male who had a welldifferentiated hepatocellular carcinoma, which is the fifth mostcommon cancer worldwide. Add prepared transfection complexes from step 3 or 4 7. General details of cell culturing and subculturing. Do you have a protocol for transfection of macrophage cell lines j774. The normal maturation process of phagosomes has been extensively studied with these macrophages 2. 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. Most cell lines can be grown using dmem culture media or rpmi culture media with 10% foetal bovine serum fbs, 2. Read the entire protocol before performing the assay. According to the literature, the cell is needed to induce by pma for 72 hours to change from monocyte to macrophage. Baseline mechanical characterization of j774 macrophages ncbi. Wash the cells precultured in a t25 flask with 5 ml of pbs and trypsinize with 1. Atcc offers custom testing services for mycoplasma detection and human cell line authentication str analysis.
Perform all steps of protocol in a laminar flow cell culture hood using sterile techniques. This information is also available as a pdf from our main menu, under customer support click here storage of frozen cells. The murine macrophagelike cell line j774 is maintained in dulbeccos modified. I am trying to use j774 macrophage for phagocytosis model. In this protocol, the mouse macrophage cell line j774a. Direct toxic effects of immunopotentiators on monocytic, myelomonocytic, and histiocytic or macrophage tumor cells in culture. The protocol for the antibiotic protection assay was carried out. Every step of the way, a wide range of cell health products maintaining healthy cells is the key to experimental success and reproducible research results. Acetaminopheninduced mitochondrial oxidative stress in. They simply do it based on passed over protocol or knowledge. J774dual cells have been derived from the mouse j774. M1 and m2 macrophage polarization in vitro with exposure. Cell culture protocols thermo fisher scientific kr. Prepare a 6well plate by adding 2 ml of j774 cell media per well.
Can anyone explain why sometimes macrophages from cell. Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. Some reagents and supplements will require filter sterilization if they are not provided sterile. Visualization of a cell with fluorescent compounds provides a wide variety of information for the analysis of cell functions. Resuspend cultures in 3 ml fresh 7h9c media2 alternatively larger culture volumes can be pelleted and concentrated into 3 ml. Please read the important technical information that follows before handling ecaccsupplied cell lines. Study of macrophage functions in murine j774 cells and. General protocol for the cryopreservation of mammalian cells. Faq id 1251 yes, please follow the protocol transfection of macrophage cell lines, including j774. Clear 96well tissueculture plate one and white 96well plate with opaque flatbottom wells two vi. Dislodge cells from the flask substrate with a cell scraper, aspirate and dispense into new flasks. The cell culture flasks are completely filled with cell culture medium to prevent loss. 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. For a 75 cm 2 flask, remove all but 10 ml of the culture medium.
Antibodydependent killing of erythrocyte and tumor targets by macrophagerelated cell lines. I am now working it by inducing the cell in flask for with pma 48 hours first add fresh medium after 24 hours, then subculture the cell by scraping the cell to 24well. Iron loading and erythrophagocytosis increase ferroportin. J774 cells, plated at a density of 10 6 cellswell, were incubated in 0. Cell culture guidelines the following is a general guideline for culturing of cell lines. Lipoprotein oxidation mediated by j774 murine macrophages. Im intending to use j774 cells to infect them with apicomplexan parasites and compare cultures in suspension vs. Below is a protocol for endpoint readings using a luminometer. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. The j774 cell lines are derived from a murine reticulum cell sarcoma whose ascites form has macrophage properties. Transfection protocol lipofectamine 3000 transfection. Pellet 3 ml of mycobacterial cultures at 2400g for 10 min. To give you confidence in the health of your cells every step of the way, weve highlighted the technologies and products within cell biology that are critical to maintaining optimal cell. The j774 cell line is grown in conventional culture medium.
To deplete j774 cells of iron, the chelators desferrioxamine mesylate dfo or salicylaldehyde isonicotinoyl hydrazone sih were added to the cell culture media to achieve final concentrations of 0 to 200. Transfer the vial contents to a centrifuge tube containing 9. J774 dual cells allow to simultaneously study the nfb pathway, by. Gcms, mpf, mtt assay, ros introduction the genus phoenix l. Optimal cell densities will vary with growth characteristics. At this point the cell lines should be subcultured or passaged in order to prevent the culture dying. We examined specific functions of macrophages phagocytic capacity, lysosomal integrity, metabolic activity, and production of reactive oxygen species ros in correlation with the cellular accumulation of the drug, using j774 mouse macrophages and thp1 human monocytes. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. To subculture the cells they need to be brought into suspension.
All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Cellbased cholesterol efflux assay kit fluorometric. The hepg2 cell line is commonly used in drug metabolism and hepatoxicity studies. C ytochrome c in both prokaryotes and eukaryotes performs a major function. Then, the same protocol described for the bacterial invasion assay was followed. Binding was determined as described under experimental. Assay for phenotype or target gene expression 48 72. A detailed method for culture of mouse macrophages can be found in. Evolution of pseudomonas aeruginosa virulence as a result.
Protocol for immunofluorescence staining of adhesion cells this is provided as a general protocol. Mammalian cell tissue culture techniques protocol abcam. Macrophages play an essential role in the early immune response to mycobacterium tuberculosis and are the cell type preferentially infected in vivo. Viral isolation protocol for cell culture introduction the optimal method for determining specific etiology of an arbovirus infection requires isolation of the virus from a specimen obtained from the patient during the acute stage of the disease and. We selected the 120 split from a few days previous. Note that cell culture conditions vary for each cell type. We therefore recommend that you familiarize yourself with your cell line of interest, and closely. The cytotoxicity of apap was evaluated by mtt cell viability and apoptosis assays. When stored properly, the kit components should remain stable for. Secure a 6cm plate of hek293t cells from the stock incubator.
The cell number and concentration determined can vary significantly depending on what method is used for counting. Both can be freezed by adding medium with 10% of glyceraldeyde and placing the vials at. While every effort is made to ensure details distributed by culture collections are accurate, culture collections cannot be held responsible for any inaccuracies in the data supplied. Wash cells briefly with hanks solution prior trypsin treatment note. Split cells under sterile conditions using basic cell culture techniques and plate approximately 1 x 105 j774.
Oritavancin, a lipoglycopeptide antibiotic in development, accumulates to high levels in the lysosomes of eukaryotic cells. All steps from this point should be carried out under strict aseptic. The j774 cell line is grown in conventional culture medium, without the addition of supplementary growth factors, resulting in nearly unlimited quantities of target cells the homogeneity of which makes this system. Cell culture we harvested bone marrow aspirates from femurs of 8 to 12week old c57bl mice. The following section describes a typical protocol to test listeriaspecific ctl lines for specific lysis of j774 cells infected with live l. Various activities and structures of a cell can be targeted for staining with fluorescent compounds fig. Primary macrophages and j774 cells respond differently to. Review and cite macrophage cell culture protocol, troubleshooting and other methodology information contact experts in macrophage cell culture to get answers.
Protocol for immunofluorescence staining of adhesion cells. The culture collections represent deposits of cultures from worldwide sources. Both reporter proteins are readily measurable in the cell culture supernatant when using quantiblue solution, a seap detection reagent. Optimization of concentration or incubation condition of the primary antibody and the secondary antibody for your own specimen is necessary. J774 murine macrophagelike cell interactions with cryptococcus. Important steps are the quenching step, the extraction and the derivatization. The following is a general guideline for culturing of cell lines. 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. Hacat and j774 cell lines were seeded in fluorodish cell culture dishes wpi. The cell culture must have 90% viability and be 80% confluent on the day of transfection. Primary macrophages and macrophagelike cell lines are commonly used as infection models, although the physiological relevance of cell lines, particularly for hostpathogen interaction studies, is debatable. Home tissue culture facility protocols general protocol for the. The viable bacteria are quantitated by the radiometric bactec method.
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