Murine bone marrow-derived macrophages constitute feeder cells for human B cell hybridomas

Hoffmann, Petra; M, Jimenez-Diaz; Weckesser, Jürgen; Bessler, Wolfgang G.

doi:10.1016/0022-1759(96)00121-4

Cited by 7 publications

(5 citation statements)

References 28 publications

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“…BMDM were differentiated in vitro from bone marrow precursor cells as described by Hoffmann et al 30 . Briefly, bone marrow cells were flushed from femurs and tibia of 6–10‐week‐old mice, washed twice in RPMI‐1640 and grown for 17 days in liquid cultures in Teflon film bags (SLG, Gauting, Germany) at 37° and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Immunostimulation by the synthetic lipopeptide P₃CSK₄: TLR4‐independent activation of the ERK1/2 signal transduction pathway in macrophages

Müller¹,

Pfannes²,

Ayoub³

et al. 2001

Immunology

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SUMMARYSynthetic lipopeptides based on bacterial lipoprotein are ef®cient activators for monocytes/ macrophages inducing the release of interleukin (IL)-1, IL-6, tumour necrosis factor-a (TNF-a), reactive oxygen/nitrogen intermediates, and the translocation of nuclear factor kB (NFkB). In this report we investigate the signal transduction pathways involved in leucocyte activation by the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P 3 CSK 4 ). We show that P 3 CSK 4 activates mitogen-activated protein (MAP)-kinases ERK1/2 and MAP kinase (MAPK)-kinases MEK1/2 in bone-marrow-derived macrophages (BMDM) and in the macrophage cell line RAW 264 . 7. Additionally, we could detect differences between the P 3 CSK 4 and lipopolysaccharide (LPS)-induced phosphorylation of MAP kinases: Different levels in phosphorylation were found both in kinetics and dose±response using RAW 264 . 7 cells or BMDM from BALB/c and LPS responder mice (C57BL/10ScSn) or LPS nonresponder mice (C57BL/10ScCr). The lipopeptide activated the MAPK-signalling cascade in both LPS responder and non-responder macrophages, whereas LPS induced the MAPK signalling pathway only in macrophages derived from LPS responder mice. An approximately 70% decrease of lipopeptide induced NFkB translocation and an about 50% reduction of nitric oxide (NO) release was observed in the presence of anti-CD14. These data correspond to the reduction of phosphorylation of ERK1/2 after stimulation with P 3 CSK 4 in the presence of anti-CD14 antibodies. Inhibition of MEK1/2 by PD98059 completely reduced the lipopeptide-induced phosphorylation of ERK1/2 indicating that MEK1/2 are solely responsible for the phosphorylation of the downstream-located MAP kinases ERK1/2.

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Section: Methodsmentioning

confidence: 99%

Immunostimulation by the synthetic lipopeptide P₃CSK₄: TLR4‐independent activation of the ERK1/2 signal transduction pathway in macrophages

Müller¹,

Pfannes²,

Ayoub³

et al. 2001

Immunology

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show abstract

“…BMDM were differentiated in vitro from bone marrow precursor cells as described [34]. Briefly, bone marrow cells were flushed from the femur and tibia of 6-10-week-old mice, washed twice in RPMI 1640 and grown in liquid cultures in teflon film bags (SLG, Gauting, Germany) at 37 jC and 5% CO 2 .…”

Section: Preparation Of Bone Marrow-derived Macrophages (Bmdms)mentioning

confidence: 99%

Triacyl-lipopentapeptide adjuvants: TLR2-dependent activation of macrophages and modulation of receptor-mediated cell activation by altering acyl-moieties

Müller¹,

Müller²,

Huber³

et al. 2004

International Immunopharmacology

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“…Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines (Grunow et al 1988). We used murine BMDM as feeder cells, a protocol proposed by us to augment both the number of obtained clones and the growth rate of CB-F7 derived hybridomas (Hoffmann et al 1996).…”

Section: Cb-f7mentioning

confidence: 99%

“…In vitro restimulation was done using rPA or rLF (List Biological Laboratories) at concentrations of 50 ng/ml in combination with PHA (2.4 g/ml) or the lipopeptide adjuvant P 3 CSK 4 (0.2-1 g/ml). B-lymphocytes were immortalized by polyethylene glycol (PEG) fusion with the humanmouse heteromyeloma cell line CB-F7 and cultivated in hybridoma medium (Biochrom) on cell culture plates (1.5 × 10 6 /ml) with bone marrow derived macrophages (1 × 10 5 /ml) as feeder cells (Hoffmann et al 1990b(Hoffmann et al , 1996. After HAT-selection, cell culture supernatants were screened for toxin specific Ig by ELISA, or for protective activity by an in vitro neutralization assay.…”

Section: Generation Of Human Monoclonal Antibodiesmentioning

confidence: 99%

Passive vaccination with a human monoclonal antibody: Generation of antibodies and studies for efficacy in Bacillus anthracis infections

et al. 2011

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Murine bone marrow-derived macrophages constitute feeder cells for human B cell hybridomas

Cited by 7 publications

References 28 publications

Immunostimulation by the synthetic lipopeptide P₃CSK₄: TLR4‐independent activation of the ERK1/2 signal transduction pathway in macrophages

Immunostimulation by the synthetic lipopeptide P₃CSK₄: TLR4‐independent activation of the ERK1/2 signal transduction pathway in macrophages

Triacyl-lipopentapeptide adjuvants: TLR2-dependent activation of macrophages and modulation of receptor-mediated cell activation by altering acyl-moieties

Passive vaccination with a human monoclonal antibody: Generation of antibodies and studies for efficacy in Bacillus anthracis infections

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Murine bone marrow-derived macrophages constitute feeder cells for human B cell hybridomas

Cited by 7 publications

References 28 publications

Immunostimulation by the synthetic lipopeptide P3CSK4: TLR4‐independent activation of the ERK1/2 signal transduction pathway in macrophages

Immunostimulation by the synthetic lipopeptide P3CSK4: TLR4‐independent activation of the ERK1/2 signal transduction pathway in macrophages

Triacyl-lipopentapeptide adjuvants: TLR2-dependent activation of macrophages and modulation of receptor-mediated cell activation by altering acyl-moieties

Passive vaccination with a human monoclonal antibody: Generation of antibodies and studies for efficacy in Bacillus anthracis infections

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Immunostimulation by the synthetic lipopeptide P₃CSK₄: TLR4‐independent activation of the ERK1/2 signal transduction pathway in macrophages

Immunostimulation by the synthetic lipopeptide P₃CSK₄: TLR4‐independent activation of the ERK1/2 signal transduction pathway in macrophages