Biochemie der Ernährung | Prof. Dr. Stefan Lorkowski - Makrophagen

Biochemie der Ernährung

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Prof. Dr. Stefan Lorkowski

 

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Biochemie der Ernährung  >>  Institut für Ernährungswissenschaften  >>  Friedrich-Schiller-Universität Jena

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Monocytic and macrophage-like cell lines

 

 

Isolating and culturing primary monocytes and macrophages for functional analyses is technically demanding and expensive. Several attempts have been made therefore to generate immortalized monocyte/macrophage-like cell lines that can be cultured easily in the amounts required for functional studies. In the following published monocyte/macrophage-like cell lines are briefly described.

 

 

ANA-1 (Mus musculus)

The ANA-1 cell line were established from bone marrow cells of C57BL/6 (H-2b) mice infected with J2 recombinant retrovirus for immortalization (Cox et al. 1989). This cell line exhibits dimorphic growth patterns with both adherent and non-adherent cells. Their morphology is characterized by numerous vacuoles and eccentric nuclei. ANA-1 cells are positive for FcyR (Ly-17), Mac-1, Ly-5 and heat stable antigen (HSA) as well as Ly5.1 and Ly6B.2 (Cox et al. 1989). These cells also express lysozyme continuously, show a very high constitutive phagocytic activity and are tumoricidally active upon LPS treatment, thus belonging to the myelomonocytic lineage. ANA-1 cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine and antibiotics (Cox et al. 1989).

 

 

AML-193 (Homo sapiens)

 

The AML-193 cell line has been established from a 13-year-old female with acute monocytic leukemia (Lange et al. 1987). Interleukin (IL)-3 and granulocyte/macrophage colony stimulating factor (GM-CSF) act synergistically to stimulate growth of these cells. Granulocyte colony stimulating factor (G-CSF) also supports short term and long term growth of AML-193 cells and acts synergistically with GM-CSF in inducing proliferation of the cells. Cultures can be maintained by addition or replacement of fresh medium (DMEM with 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 5 ng/ml GM-CSF and 5% fetal bovine serum). The cultures should be started at a density of 3 x 105 cells/ml and maintained between 3 x 105 and 1 x 106 cells/ml. The cells require GM-CSF for long term growth. Conditioned medium from U-87MG cells can be used at a concentration of 10% to replace GM-CSF in the culture medium (Santoli et al. 1987).

 

 

C7 (Mus musculus)

 

The C7 cell line has been established from bone marrow of male adult 129/Sv p53-/- mice (Miyamoto et al. 1998). The cells share the characteristics of their cell surface molecules and phagocytotic activity with macrophages. The differentiation potential of C7 cells to osteoclasts is maintained in the presence of M-CSF and abolished by treatment with lipopolysaccharide (LPS) that is known to activate macrophages. The C7 cell line can be cultured in alpha-MEM containing 10% fetal bovine serum and 1 ng/ml human or murine M-CSF. After reaching confluence cells can be divided 1:2 to 1:4 every four to six days using trypsin/EDTA after washing twice with PBS. C7 cells have a doubling time of about 80 to 100 hours.

 

 

DH82 (Canis familiaris)

 

The DH82 cell line was established from neoplastic progenitor cells of canine malignant histiocytosis of a ten year old male Golden Retriever (Wellman et al. 1988). The cells have a macrophage-like morphology and are able to phagocytose latex particles. They are positive for Fc-γ receptors and negative for Fc-µ and C3b receptors. However, DH82 cells do not produce IL-1. They can be cultured with Eagle's Minimum Essential Medium (EMEM) supplemented with 2 mM glutamine, 1% non essential amino acids and 15% fetal bovine serum. Subcultures are prepared by treating cells with 0.25% trypsin/0.03% EDTA solution at 37°C. A subcultivation ratio of 1:4 to 1:8 is recommended.

 

 

DMBM-2 (Mus musculus)

 

The DMBM-2 cell line has been established from bone marrow of a female C3H/HeJ mouse (Monner et al. 1997). The macrophages grow in aggregates, mostly loosely adherent, and some of them have long processes. The cells can be cultured in Roswell Park Memorial Institute (RPMI)-1640 containing 20% horse serum, 1 mM sodium pyruvate, 1 mM non-essential amino acids, 1.25 µg/ml vitamin B12, 75 µM α-thioglycerin, 2 mM L-glutamine and 2.5% 10x L-929 supernatant. Cells can be split 1:10 every four to six days by detaching the cells by flushing adherent cells with medium through a 25G needle. Cells grow best in round Corning plates and have a doubling time about 16 to 22 hours.

 

 

GG2EE (Mus musculus)

 

GG2EE cells were isolated from C3H/HeJ mice. Immortalization was achieved by infection with J2 recombinant retrovirus (Blasi et al. 1987). Cells grow in suspension but may also adhere loosely to culture vessels (Cox et al. 1989). This cell line belongs to the macrophage lineage and is positive for MAC-1, MAC-2 and Fc receptor (Blasi et al. 1987). In addition these cells express Ly-5.1 antigens as well as M169 and F4/80 antigens, but they do not show any tumoricidal activity upon stimulation with LPS (Blasi et al. 1987). GG2EE cells are cultivated in DMEM supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine and antibiotics (Cox et al. 1989).

 

 

HD11 (Gallus gallus)

HD11 cells were derived drom chicken bone marrow cells after transformation with MC29 virus (avian leukemia virus) (Beug et al. 1979). The morphology of these cells is amoeboid, often with polar shape and a diameter of 15-20 µm. The cells are slightly adherent in culture and display numerous lamellipodia. The nucleus is located eccentrically and the cytoplasm contains granules and vacuoles (Beug et al. 1979). HD11 cells are macrophage-like in morphology and functionality and show phagocytic activity. These cells are positive for Fc receptor and Mac antigen (Beug et al. 1979). HD11 cells are maintained in DMEM supplemented with 8 % FCS, 2 % chicken serum and 10 mM HEPES (Beug et al. 1979).

 

 

HL-60 (Homo sapiens)

 

HL-60 is a promyelocytic cell line derived by Collins et al. in 1977. Peripheral blood leukocytes were obtained by leukopheresis from a 36-year-old Caucasian female with acute promyelocytic leukemia. HL-60 cells differentiate spontaneously but differentiation can be also stimulated by butyrate, hypoxanthine, phorbol-12-myristate-13-acetate, 12-O-tetradecanoylphorbol-13-acetate, dimethylsulfoxide (DMSO), actinomycin D and retinoic acid (Collins et al. 1978). The cells exhibit phagocytic activity and responsiveness to chemotactic stimuli (Gallagher et al. 1979). The HL-60 cell line is positive for myc oncogene expression. Cultures can be maintained by addition of fresh medium or replacement of medium (RPMI-1640 containing 10% fetal calf serum) every two to three days depending on cell density. Alternatively, cultures can be established by centrifugation with subsequent resuspension in fresh medium at a cell density of 5 x 106 cells/ml. Cultures are maintained at cell concentrations between 5 x 105 and 2 x 106 cells/ml.

 

 

IC-21 (Mus musculus)

 

The IC-21 cell line was obtained by transformation of normal C57BL/6 mouse peritoneal macrophages with SV40 (Mauel et al. 1971). This line shares many properties with normal mouse macrophages and displays macrophage specific antigens. IC-21 cells have phagocytic and cytolytic properties, can lyse tumor targets in vitro and appear to be more differentiated than cells of the P388D1 macrophage line (Walker et al. 1975 and 1980). The cells produce large quantities of acid so RPMI-1640 containing 10% fetal calf serum should be changed at least three times per week. The cell monolayer should be rinsed with 10 to 15 ml of calcium and magnesium free PBS and an additional 10 to 15 ml of the same solution. The culture should be stood for 5 to 10 minutes at room temperature. The flask is then struck to dislodge the cells and 5 to 7 ml of the cell suspension are added to a flask containing less than 10 ml of growth medium. Once the cells have attached (after one to two days) additional medium should be added. Subculturing is necessary when cells became confluent. A subcultivation ratio of 1:2 to 1:4 is recommended.

 

 

INF-3A (Mus musculus)

 

INF-3A cells were derived from bone marrow of C3H/HeN (H-2k) mice (Cox et al. 1989). Immortalization was performed by infection with J2 recombinant retrovirus. Cells grow in suspension with some cells loosely adherent. INF-3A cells are characterized by a large nuclear to cytoplasmic ratio and few cytosolic vacuoles. This cell line is positive for FcyR (Ly-17), Mac-1, Ly-5 and heat-stable antigen (HAS) (Cox et al. 1989). INF-3A cells also expresses lysozyme and show phagocytic activity, which places this cell line into the myelomonocytic lineage (Cox et al. 1989). Upon stimulation with LPS these cells have shown tumoricidal activity. INF-3A cells are maintained in DMEM supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine and antibiotics (Cox et al. 1989).

 

 

J774A.1 (Mus musculus)

 

J774 tumor arose in a female BALB/c/NIH mouse with a reticulum cell sarkoma during a plasmacytoma induction program in 1968 (Hirst et al. 1971). The initial tumor of which the cells designated as J774.A1 had been isolated was ascitic. J774A.1 macrophages are active in antibody dependent phagocytosis (Ralph et al. 1975). Their growth is inhibited by dextran sulfate, p-phenylendiamin and LPS. J774.A1 cells synthesize large amounts of lysozyme and exhibit minor cytolysis but predominantly antibody-dependent phagocytosis. Interleukin 1β is synthesized continuously by this cell line (Ralph et al. 1977b). J774.A1 cells have a doubling time of 17 hours and can be cultured under the same conditions as RAW 264.7 macrophages.

 

 

J2-BM (Mus musculus)

The J2-BM cell line was obtained from femurs of C3H/HeJ mice, whose B-cell and macrophage response to LPS is genetically knocked out (Blasi et al. 1985). For immortalization cells were infected with J2 recombinant retrovirus and the cells were found to express v-myc and v-raf. J2-BM cells are a non-adherent, monocytic cell line characterized by the expression of lysozyme and nonspecific esterase (Blasi et al. 1985). They are positive for MAC-1 and Fc receptors as well as mouse heat-stable antigen, asialo-GM1 glycolipid and F4/80 antigen. Propagation of J2-BM cells requires the presence of dextran beads (Blasi et al. 1985).

 

 

Kasumi-3 (Homo sapiens)

 

The Kasumi-3 cell line was established from blast cells of a 57-years-old male myeloperoxidase-negative acute leukemia patient from Japan (Asou et al. 1996). Kasumi-3 cells express CD7, CD4, CD13, CD33, CD34, HLA-DR and c-Kit on their cell surface, a finding that is compatible with that of acute myelocytic leukemia, subtype M0 (AML-M0). Kasumi-3 cells have t(3;7)(q27:q22), del(5)(q15), del(9)(q32), and add(12)(p11) chromosomal abnormalities. The breakpoint of 3q27 is located near the EVI1 gene and high levels of expression of the EVI1 gene were observed. Kasumi-3 cells treated with 12-O-tetradecanoylphorbol-13-acetate show maturation of the monocytic lineage. Treatment with either IL-2, IL-3, IL-4, GM-CSF or stem cell factor induces proliferation, a characteristic of undifferentiated leukemia. Kasumi-3 cultures can be maintained by the addition of fresh medium or replacement of the medium. Alternatively, cultures can be established by centrifugation with subsequent resuspension at a cell density of 3 x 10cells/ml. Depending on cell density (between 3 x 10and 3 x 106 cells/ml) the medium should be renewed every two to three days.

 

 

M1 (Mus musculus)

 

The M1 cell line was established in 1969 in vitro from spontaneous myeloid leukemia of SL strain mice (Ichikawa et al. 1969). The cells can be induced to become macrophage-like by a variety of stimulating agents including dexamethasone, LPS and WEHI-3 conditioned medium (Ralph et al. 1983). M1 cultures can be maintained by addition or replacement of fresh RPMI-1640 medium containing 10% fetal calf serum. Cultures are started at a cell density of 1 x 105 cells/ml and maintained between 1 x 105 cells/ml and 1 x 106 cells/ml. Medium has to be renewed every two to three days.

 

 

Mono Mac 6 (Homo sapiens)

 

The monocytic cell line Mono Mac 6 was established from peripheral blood of a 64-year-old man with relapsed acute monocytic leukemia (AML FAB M5) in 1985 following myeloid metaplasia. The cells were assigned to the monocyte lineage on the basis of morphological, cytochemical and immunological criteria. Mono Mac 6 cells express sodium fluoride sensitive non-specific esterase, produce reactive oxygen and stain with monoclonal antibody My4. The cells constitutively exhibit phagocytosis of antibody-coated erythrocytes in 80% of the cells and react with a panel of monoclonal antibodies that are specific for mature monocytes (i.e. M42, LeuM3, 63D3, Mo2 and UCHMI). By contrast, the monoblastic cell lines U937 and THP-1 are negative for all these markers. Only expression of My4 could be detected after differentiation induced by interferon-γ. In addition, the cells show an increased frequency of multinucleated cells with a rise from 4.8% to 21.9%. Mono Mac 6 cells are negative for CD3, CD19, and CD34, and express CD13, CD14, CD15, CD33, CD68, and HLA-DR. The cells are single and round (multi-formed) or form small clusters in suspension; they are sometimes loosely adherent. One to five percent of the cells are giant cells. The Mono Mac 6 cell line was tested negative for reverse transcriptase, EBV, HBV, HCV, HHV-8, HIV, HTLV-I/II and is cultured in RPMI 1640 containing 10% fetal bovine serum, 2 mM L-glutamine, non-essential amino acids, 1 mM sodium pyruvate and 9 µg/ml bovine insulin. Cells are maintained at 0.3 to 1.0 x 106 cells/ml; the split ratio is 1:3 to 1:5. They are seeded out at about 0.3 x 106 cells/ml at 37°C with 5% CO2 and a maximal density at about 1.0 x 106 cells/ml. Their doubling time is about 60 hours.

 

 

MH-S (Mus musculus)

 

The MH-S cell line was derived in 1989 by SV40 transformation of an adherent cell-enriched population of mouse alveolar macrophages from a seven-weeks-old male (Mbawuike & Herscowitz 1989). The cells retain many of the properties of alveolar macrophages including typical macrophage morphology. They are adherent, phagocytic, esterase positive and peroxidase negative. LPS treatment stimulates IL-1 production. The cells are capable of suppressing the in vitro plaque forming cell response in a cell-dose-dependent manner (Mbawuike & Herscowitz 1989). For culturing MH-S cells RPMI-1640 medium supplemented with 10% fetal calf serum and 0.05 mM 2-mercaptoethanol is used. Cultures can be maintained by transferring the floating cells to a centrifuge tube. Adherent cells are rinsed with 0.25% trypsin/0.53 mM EDTA solution. The solution is removed and an additional 1 to 2 ml of trypsin/EDTA solution is added. Incubation at room temperature (or at 37°C) is performed until the cells detach. The detached cells are added to the floating cells collected as described above and the cell suspension is centrifuged at 1,000 rpm for ten minutes. The cell pellet is resuspended in fresh medium, aspirated and dispensed into new flasks. A subcultivation ratio of between 1:2 to 1:6 is recommended and the medium has to be replaced every two to three days.

 

 

MV-4-11 (Mus musculus)

 

The MV4-11 cell line was established by Rovera and colleagues in 1987 from the blast cells of a ten-year-old male with biphenotypic B-myelomonocytic leukemia (Lange et al. 1987). The growth factor, granulocyte/macrophage colony-stimulating factor (GM-CSF), was required to establish this cell line and growth factors are necessary for its continuous proliferation in chemically defined medium (Lange et al. 1987). However, this cell line can be propagated in medium (Iscove's Modified Dulbecco's Medium) supplemented with 10% fetal bovine serum without the addition of growth factors. To maintain the cells in serum-free medium, it is necessary to add 0.005 mg/ml transferrin, 0.005 mg/ml insulin and 5 ng/ml GM-CSF. Interleukin-3 independently supports the long-term growth of this cell line but IL-3 also antagonizes the proliferation of MV4-11 cells in the presence of GM-CSF when both factors are used at very low concentrations. Granulocyte colony stimulating factor synergizes with GM-CSF in inducing the proliferation of MV4-11 cells. Granulocyte colony stimulating factor alone causes a transient stimulation of these cells. More than 96% of MV4-11 cells are positive by indirect immunofluorescence for the myelomonocytic antigen CD15, 40-96% are positive for the monocytic antigen CD4 and 4-11% are positive for CD10 (Santoli et al. 1987). Cultures of MV4-11 cells can be maintained by adding or replacing fresh medium as described above. Cultures are started at a cell density of 2 x 105 cells/ml and maintained between 1 x 105 and 1 x 106 cells/ml. The medium is to be renewed every two to three days.

 

 

P388.D1 (Mus musculus)

 

The murine P-388D1 monocyte/macrophage cell line has been established in 1975 from the ascites of a DBA/2 mouse with a lymphoid neoplasm (Koren et al. 1975). P-388D1 cells are elliptical and round loosely adherent cells growing in monolayers. They produce IL-1 and LAF (lymphocyte activating factor) after stimulation with LPS or phorbol-12-myristate-13-acetate. The cells phagocytose zymosan and latex beads and are active in antibody dependent cell-mediated cytotoxicity systems. They are negative for surface immunoglobulin and can be cultured with RPMI-1640 medium containing 10% fetal bovine serum. Subcultures are prepared by scraping. For this purpose, the old medium is removed, fresh one is added and the cells are dislodged. The cells are dispensed into new flasks with a subcultivation ratio of between 1:4 to 1:8. The medium has to be renewed three times per week.

 

 

PMJ2-R (Mus musculus)

 

PMJ2-R cells were derived from murine peritoneal macrophages (Adami et al. 1993). Immortalization was achieved by infection with the J2 virus. The obtained cell line shows oval or round cells with a basophilic cytoplasm, numerous vacuolae and an oval or kidney-shaped nucleus (Adami et al. 1993). PMJ2-R cells are positive for vraf, Mac-1, Mac-2, Fc receptor, Ly-5.1, F4/80 as well as CSF1 receptor (Adami et al. 1993). On the other hand these cells were found to be negative for Lyt-1.2 and Thy-1.2. Furthermore PMJ2-R cells exhibit phagocytotic activity and esterase activity. Upon a LPS and IFNγ stimulus these cells show functional cytotoxicity and secrete IL-6 (Adami et al. 1993). PMJ2-R cells are maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5 mM HEPES (pH 7.4), 4 mM L-glutamine, 50 µg/ml gentamicin, 4 µg/ml fungizone and 5 % FBS (Palleroni et al. 1991).

 

 

PU5-1.8 (Mus musculus)

 

PU5-1.8 macrophages emenate from a BALB/c mouse lymphoid tumor (Ralph et al. 1976). The cells have immunoglobulin and complement receptors, are resistant to dextran sulfate and produce isoenzyme. Further, they are capable of antibody-dependent lysis of sheep erythrocytes and tumor targets. The cell line is sensitive to growth inhibition by LPS and purified protein derivative (PPD) from Mycobacteria (Ralph & Nakoinz 1977a). They can be cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10% horse serum. The cell density should be between 3 to 9 x 100,000 cells/ml. Cells can be detached by aspirating or gentle shaking.

 

 

R2 (Rattus norvegicus)

 

Rat R2 macrophages were originally isolated after silica injection into pleural cavity described by J.J. Harvey et al. (unpublished according to van den Berg et al. 1996). The cells are maintained in RPMI 1640 with 20 mM Hepes and 10 % FCS, 2 mM glutamine and pencillin streptomycin and amphotericin B (Hauton et al. 2002). Alternatively cell culture using only 100 U/ml pencillin and 100 pg/ml streptomycin without amphotericin B is possible as well (van den Berg et al. 1996). Rat R2 macrophages should be subcultured at 80 % confluence, the cells can be detached using trypsin/EDTA and remain viable for 3-8 passages, 4 h after subcultivation the medium is exchanged to remove any non-adherent cells (Hauton et al. 2002).

 

 

RAW 264.7 (Mus musculus)

 

The RAW 264.7 male mouse macrophage cell line was established from a tumor induced by Abelson murine leukemia virus (MuLV). The cell line was first reported by Raschke et al. in 1976. RAW 264.7 cells are negative for surface immunoglobulin, and surface antigens Ia and Thy-1.2. This cell line was reported not to secrete detectable amounts of viruses. However, unpublished data communicated by Janet W. Hartley indicates the expression of infectious ecotropic Abelson MuLV closely related, if not identical, to the Moloney MuLV helper virus used in the original virus inoculum. The cells also express polytropic MuLV, unsurprisingly based on the mouse passage history of the virus stocks. The cells pinocytose neutral red and phagocytose latex beads and zymosan. They are capable of antibody dependent lysis of sheep erythrocytes and tumor cell targets. Lipopolysaccharide or p-phenylendiamin treatment for two days stimulates lysis of erythrocytes but not tumor cell targets (Ralph et al. 1977a). It is of note that RAW 264.7 cells are no monocytes but dividing macrophages. RAW 264.7 cells are cultured in DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplemented with 10% fetal bovine serum. Subcultures are prepared by dislodging the cells from the flask substrate with a cell scraper, aspirating and adding appropriate aliquots of the cell suspension into new culture vessels. 

 

 
THP-1 (Homo sapiens)

 

The THP-1 cell line, first described by Tsuchiya et al. in 1980, was isolated from the blood of a one year old, likely Japanese boy with acute monocytic leukemia. These monocytes have Fc and C3b receptors, but no surface or cytoplasmic immunoglobulins. HLA haplotypes of THP-1 cells are HLA-A2, -A9, -B5, -DRW1 and -DRW2. The monocytic nature of the cell line is characterized by: (i) the presence of alpha-naphthyl butyrate esterase activities which can be inhibited by sodium fluoride, (ii) lysozyme production, (iii) the phagocytosis of latex particles and sensitized sheep erythrocytes, and (iv) the ability to restore T-lymphocyte response to Concanavalin A. The cells do not possess Epstein-Barr virus-associated nuclear antigen (Tsuchiya et al. 1980). They have a doubling time of approximately 30 hours and are cultured in RPMI-1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1.0 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol and 10% fetal bovine serum. Monocytic differentiation of THP-1 cells into macrophages can be induced using phorbol esters such as phorbol-12-myristate-13-acetate. After four days of phorbol ester treatment, THP-1 cells are matured into macrophages.

 

 

TUR (Homo sapiens)

 

TUR (TPA-U937-Resistant) is a stably transfected cell line generated by Ralf Hass and Masanori Hirano in 1992 from the U-937 cell line. U-937 cells were transfected by electroporation with the pMG2neoPA plasmid containing the neomycin resistant gene and then selected in medium containing G418 and 12-O-tetradecanoylphorbol-13-acetate (TPA) (Hass et al. 1993). TUR cells have remained resistant to both G418 and 12-O-tetradecanoylphorbol-13-acetate after culture in the absence of these agents for over 100 passages. The cell line retains monocytic properties but does not differentiate along the macrophage pathway by phorbal ester treatment. While treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate is associated with growth arrest and induction of monocytic differentiation, the TUR cell line is unresponsive to the growth-inhibitory effects of this agent. Cultures can be maintained by addition of fresh medium every two to three days depending on the cell density or replacement of medium (RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-glutamate). Alternatively, cultures can be established by centrifugation with subsequent resuspension in fresh medium at 5 x 106 cells/ml. Cultures are maintained at cell concentrations between 5 x 105 and 2 x 106 cells/ml.

 

  

U-937 (Homo sapiens)

 

The U-937 cell line was derived by Kenneth Nilsson and Crister Sundstrom in 1974 from malignant cells obtained from the pleural effusion of a 37 years old male Caucasian patient with histiocytic lymphoma (Sundstrom et al. 1976). Studies since 1979 have shown that U-937 cells can be induced to terminal monocytic differentiation by supernatants from human mixed lymphocyte cultures, phorbol esters, vitamin D3, interferon-γ, tumor necrosis factor-α and retinoic acid. In 1994, PCR and cytogenetic analyses showed that a number of stocks of U-937 were contaminated with the human myeloid leukemia cell line K-562. A stock of U-937 was found to be free of K-562. K-562 free preparations are now offered as authentic U-937 cells (Reid et al. 1995). U-937 cells are cultured under the same conditions as THP-1 cells.

 

 

WEHI-3 (Mus musculus)

 

The WEHI-3 cell line was derived from monocytes of a murine leukemia model in 1976 (Ralph et al. 1976). The cells have a macrophage-like morphology and produce IL-3, granulocyte colony stimulating factor and lysozyme (Ralph & Nakoinz 1977a). The growth of WEHI-3 is inhibited by 4 ng/ml LPS and 30 to 40 mcg/ml dextran sulfate and is blocked by higher concentrations of LPS (Ralph & Nakoinz 1974). Latex beads are phagocytized but are not toxic. Zymosan and Bacille Cakmette-Guérin are phagocytized and block growth of WEHI-3 cells. The cells exhibit only weak effector activity in antibody-dependent cell-mediated cytotoxicity (Ralph & Nakoinz 1977b). Cultures can be maintained by the addition of fresh medium or replacement of medium. Alternatively, cultures can be established by centrifugation with subsequent resuspension at 2 x 105 cells/ml. Iscove's modified Dulbecco's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 0.05 mM 2-mercaptoethanol and 10% fetal bovine serum is recommended.

 

 

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Stefan Lorkowski | 2008-2011

 

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