Optimal conditions for the generation of monoclonal antibodies using primary immunisation of mouse splenocytes in vitro under serum-free conditions

1989 ◽  
Vol 121 (2) ◽  
pp. 253-260 ◽  
Author(s):  
Mark De Boer ◽  
Ferry A. Ossendorp ◽  
Gert Van Duijn ◽  
Gerald H.J. Ten Voorde ◽  
Joseph M. Tager
Keyword(s):  
Monoclonal Antibodies ◽  
Optimal Conditions ◽  
Mouse Splenocytes ◽  
Serum Free ◽  
Primary Immunisation ◽  
Serum Free Conditions

DETECTION OF INACTIVATED α2-MACROGLOBULIN (α2M) IN HUMAN PLASMA USING MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
J Abbink ◽  
J Nuijens ◽  
C Huijbregts ◽  
E Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Longitudinal Studies ◽  
Human Plasma ◽  
Dextran Sulfate ◽  
Optimal Conditions ◽  
Α2 Macroglobulin ◽  
In Vitro Activation

Monoclonal antibodies (mAbs) were raised against human a2M. Five mAbs that bound to α2M in ELISA were further analyzed by a radioimmunoassay (RIA) for their reaction with three types of α2M: native α2M, chemically inactivated α2M (iα2M) (methylamine treated), and proteolytically iα2M. One mAb reacted with all forms of α2M, while four mAbs bound both forms of ia2M but not native α2M. One of these latter mAbs (Ml) was used to develop a RIA (the Ml-assay) for the detection of iα2M in plasma: Ml coupled to Sepharose is incubated with the plasma to be tested, and bound iα2M is detected by a subsequent incubation with polyclonal 125I-anti-α2M antibodies. As little as 5 ng of iα2M can be detected with this assay in the presence of an excess of native α2M. This assay was then applied to measure inactivation of α2M in vitro and in vivo. In vitro activation of the contact system in plasma by dextran sulfate results in the inactivation of ca 10% of α2M. When blood from normal donors was collected under optimal conditions, about 0.5% of the total α2M content appeared to be iα2M. Longitudinal studies in patients (a.o. with septicaemie, during cardiopulmunary bypass) revealed that increased levels of iα2M occurred sporadically. The Ml-assay appears to be useful to monitor the role of α2M in human diseases.

scholarly journals Defined, serum-free conditions for in vitro culture of primary human T-ALL blasts

Leukemia ◽  
2012 ◽  
Vol 27 (6) ◽  
pp. 1437-1440 ◽  
Author(s):  
A J Yost ◽  
O O Shevchuk ◽  
R Gooch ◽  
S Gusscott ◽  
M J You ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Serum Free ◽  
Serum Free Conditions

scholarly journals Evidence for direct action of human biosynthetic (recombinant) GM-CSF on erythroid progenitors in serum-free culture

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1867-1871 ◽  
Author(s):  
AR Migliaccio ◽  
M Bruno ◽  
G Migliaccio
Keyword(s):  
Direct Action ◽  
Erythroid Progenitors ◽  
Serum Free ◽  
Gm Csf ◽  
Plastic Surface ◽  
Blast Cells ◽  
Fetal Bovine ◽  
Serum Free Conditions ◽  
Stimulating Factor

Abstract The biologic activity of human biosynthetic granulocyte-monocyte colony stimulating factor (GM-CSF) was investigated in serum-free culture of erythroid progenitors derived from adult peripheral blood. The morphology of erythroid bursts and the cloning efficiency of BFU-E under serum-free conditions were similar to those observed in dishes with fetal bovine serum (FBS). For these experiments, progenitor cells were partially purified by Ficoll-Paque density centrifugation, adherence to a plastic surface, and complement-mediated cytotoxicity of Leu-1+ elements. For some studies, blastlike cells were harvested directly from 6-day-old semisolid cultures. In serum-free culture of the light-density cell fraction, biosynthetic erythropoietin (Ep) was sufficient for formation of pure and mixed erythroid colonies whereas GM-CSF was required for granulocyte-monocytic colonies. When adherent and Leu-1+ cells were removed, or when in vitro differentiated blast cells were used as a source of progenitors, neither Ep or GM-CSF alone induced colony formation. In dishes supplemented with both growth factors, erythroid bursts were detected. Although the presence of GM- CSF alone did not induce formation of any colony or clusters, BFU-E were recorded when Ep was added 8 days later, suggesting that BFU-E could be maintained. Terminal maturation of the resulting erythroid bursts was delayed by 8 days. These results provide evidence that GM- CSF acts directly on early erythroid progenitors. Furthermore, they suggest that both Ep and GM-CSF are necessary to start the differentiation process.

scholarly journals Effects of C-Mpl Ligand on Cytoplasmic Maturation of Murine Megakaryocytes and on Platelet Production

1998 ◽  
Vol 46 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Yoji Ishida ◽  
Toshiharu Ito ◽  
Shin-ichiro Kuriya
Keyword(s):  
Progenitor Cells ◽  
Biological Effects ◽  
Primary Factor ◽  
Serum Free ◽  
Platelet Production ◽  
Nuclear Development ◽  
Cytoplasmic Maturation ◽  
Serum Free Conditions ◽  
Ache Expression

To test the hypothesis that the c-mpl ligand is not a primary factor in thromb-ocytopoiesis, we investigated the biological effects of recombinant human (rh) c-mpl ligand on differentiation of murine progenitor cells and on maturation of the cultured murine megakaryocytes under serum-free conditions on the basis of ploidy distribution, megakaryocyte/platelet-specific surface antigen CD 61 [glycoprotein (GP) IIIa], and cytoplasmic acetylcholinesterase (AchE) expression in vitro. In addition, we studied the effect of c-mpl ligand on proplatelet formation (PPF) by murine mature megakaryocytes. AchE was less strongly expressed in cultured megakaryocytic cells stimulated by c-mpl ligand than in those stimulated by recombinant murine (rm) IL-3 + rh IL-6 during the differentiation of progenitor cells. Less CD 61 was expressed by c-mpl ligand during both the differentiation of progenitor cells and the maturation of megakaryocytes compared with that by rm IL-3 + rh IL-6. Endomitosis, however, was more stimulated by c-mpl ligand than by rm IL-3 + rh IL-6 under both conditions. Furthermore, PPF of mature megakaryocytes was not stimulated by c-mpl ligand. These results indicate that c-mpl ligand stimulates the nuclear development of megakaryocytic cells but that it does not stimulate cytoplasmic maturation and PPF as much as IL-6. These data strongly suggest that c-mpl ligand is not a primary factor in platelet production.

190 EFFICIENT GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM PORCINE ADIPOSE-DERIVED STEM CELLS WITH A FEEDER-INDEPENDENT AND SERUM-FREE SYSTEM

2014 ◽  
Vol 26 (1) ◽  
pp. 209
Author(s):  
Y. Zhang ◽  
C. Wei ◽  
P.-F. Zhang ◽  
X. Li ◽  
Y.-S. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Adipose Derived Stem Cells ◽  
Free System ◽  
Serum Free ◽  
Induced Pluripotent ◽  
Serum Free Conditions

Somatic cells could be directly reprogrammed into stem state by ectopic expression of transcription factors, which share similar features of embryonic stem cells (ESC). Induced pluripotent stem cells (iPSC) possess promising application in producing genetically modified animals, whereas the generation of porcine offspring from iPSC is still difficult and controversial, and new materials are needed. In this study, we report the generation of iPSC from porcine adipose-derived stem cells (pADSC) using drug-inducible expression of defined human factors (Oct4, Sox2, Klf4, and c-Myc) and ‘2i’ plus leukemia inhibitory factor (LIF) culture system. pADSC were isolated from subcutaneous adipose tissue of a 28-day-old Danish Landrace, and subsequently characterised by high proliferation rate at low passages, long period passaging without significant replication senescence, mesenchymal stem cell-specific surface markers expression, including CD29 (0.995 ± 0.0577), CD44 (0.999 ± 0.0333), and CD90 (0.994 ± 0.0333), together with successful adipogenic and osteogenic differentiation ability in vitro. The reprogramming of iPSC from pADSC was evidently more efficient than the process from adult fibroblasts (P < 0.01), both of which were carried out under feeder-independent and serum-free conditions, and this may be due to the higher demethylation level of genomic DNA in pADSC. Two lines of porcine iPSC with naïve-like state were finally obtained through feeder-independent and serum-free conditions. The successful reprogramming of iPSC was demonstrated by short cell cycle interval, alkaline phosphatase (AP) staining positive, expression of stemness-related proteins including OCT-4, SOX2, NANOG, SSEA3, and SSEA4. Full reprogramming of iPSC was evaluated by the significant up-regulation of LIN28, ESRRB, UTF1, and DPPA5. Naïve-like state of porcine iPSC was further confirmed by the striking resemblance to naïve mESC, single-cell dissociation, LIF-dependency, up-regulation of STELLA and ERAS, and little translation of TRA-1-60 and TRA-1-81. In addition, porcine naïve-like iPSC possessed normal karyotypes, and could differentiate into cell types of all three germ layers in vitro and in vivo. Furthermore, in vivo studies to determine the capacity of these cells to integrate into the inner cell mass of blastocysts are still being undertaken for validation. Together, our study provided an efficient method to derive porcine naïve-like iPSC from pADSC, which may be useful for the production of living offspring. Y. Zhang and C. Wei contributed equally to this work. Y.-H. Zhang is the corresponding author. This work was supported by the National Natural Science Foundation Program 31272442.

Osteogenic protein-1 promotes growth and maturation of chick sternal chondrocytes in serum-free cultures

1995 ◽  
Vol 108 (1) ◽  
pp. 105-114 ◽  
Author(s):  
P. Chen ◽  
S. Vukicevic ◽  
T.K. Sampath ◽  
F.P. Luyten
Keyword(s):  
Bone Morphogenetic Protein ◽  
Chondrocyte Differentiation ◽  
Mrna Levels ◽  
Embryonic Chick ◽  
Serum Free ◽  
Chondrocyte Maturation ◽  
Morphogenetic Protein ◽  
Osteogenic Protein ◽  
Serum Free Conditions

We examined the effect of recombinant human osteogenic protein-1 (OP-1, or bone morphogenetic protein-7), a member of the bone morphogenetic protein family, on growth and maturation of day 11, 15 and 17 chick sternal chondrocytes in high density monolayers, suspension and agarose cultures for up to 5 weeks. OP-1 dose-dependently (10-50 ng/ml) promoted chondrocyte maturation associated with enhanced alkaline phosphatase activity, and increased mRNA levels and protein synthesis of type X collagen in both the presence and absence of serum. In serum-free conditions, OP-1 promoted cell proliferation and chondrocyte maturation, without requiring either thyroid hormone or insulin, agents known to support chick chondrocyte differentiation in vitro. When grown in agarose under the same conditions, TGF-beta 1 and retinoic acid neither initiated nor promoted chondrocyte differentiation. The results demonstrate that OP-1, as the sole medium supplement, supports the maturation of embryonic chick sternal chondrocytes in vitro.

scholarly journals Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine.

1992 ◽  
Vol 116 (4) ◽  
pp. 1035-1042 ◽  
Author(s):  
K Böhme ◽  
M Conscience-Egli ◽  
T Tschan ◽  
K H Winterhalter ◽  
P Bruckner
Keyword(s):  
Growth Factor ◽  
Culture Media ◽  
Insulin Like Growth Factor ◽  
Serum Free ◽  
Matrix Deposition ◽  
Chondrocyte Maturation ◽  
Factor I ◽  
Growth Factor I ◽  
Serum Free Conditions

In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.

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