Hybridoma antibody production in vitro in type II serum-free medium using Nutridoma-SP supplements Comparisons with in vivo methods

1991 ◽  
Vol 145 (1-2) ◽  
pp. 213-221 ◽  
Author(s):  
Geneviève Federspiel ◽  
Kenneth C. McCullough ◽  
Ulrich Kihm
Keyword(s):  
Antibody Production ◽  
Type Ii ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free

scholarly journals The role of neutrophil membrane glycoprotein GP-150 in neutrophil adherence to endothelium in vitro

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 167-178 ◽  
Author(s):  
JM Harlan ◽  
PD Killen ◽  
FM Senecal ◽  
BR Schwartz ◽  
EK Yee ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Membrane Glycoprotein ◽  
Ionophore A23187 ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Endothelial Monolayers ◽  
Neutrophil Adherence

Abstract We have previously described two patients with a congenital defect in neutrophil function characterized by an inability to form pus. The patients' neutrophils lack a membrane glycoprotein of mol wt 150,000 daltons (GP-150) on analysis by SDS-PAGE. This glycoprotein is part of a membrane antigen complex recognized by the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produces defects in chemotaxis and phagocytosis in vitro similar to those observed in the patients. Since neutrophil adherence to vascular endothelium is prerequisite to neutrophil emigration in vivo, we examined the interaction of the patients' neutrophils and normal neutrophils treated with MoAb 60.3 with cultured endothelium. Adherence was determined as the percentage of 51Cr-labeled purified peripheral blood neutrophils which remained adherent to plastic wells or endothelial monolayers after a 45-minute incubation at 37 degrees C. The percentage of neutrophils from patient 1 remaining adherent to uncoated, fibronectin-coated, or laminin-coated plastic was similar to that observed in normal neutrophils (55% to 84% adherence with normal neutrophils v 73% to 78% adherence with the patient's neutrophils and 63% to 82% adherence with MoAb 60.3-treated normal neutrophils). The adherence of the neutrophils from patient 1 and MoAb 60.3-treated normal neutrophils to human or bovine endothelium in serum-free medium was also not significantly different from that observed in normal neutrophils (less than 10% adherence with normal, MoAb 60.3-treated, and patient neutrophils). In medium containing 10% autologous or heterologous human plasma, however, the adherence of neutrophils from patient 1 or MoAb 60.3-treated normal neutrophils to endothelial monolayers was significantly reduced (35% +/- 7% of normal neutrophils in seven experiments). Although phorbol myristate acetate (PMA) (10 ng/mL) and calcium ionophore A23187 (10(-5) mol/L) markedly increased the adherence of normal neutrophils to endothelial monolayers in serum- free medium (40% to 85% adherence), neither agent increased the adherence of the neutrophils from patient 1 or normal neutrophils treated with MoAb 60.3 (less than 5% adherence). The adherence of PMA- activated neutrophils from patient 2 to endothelial monolayers was also markedly decreased when compared with that of normal neutrophils. Postsecretory cell-free supernatants from PMA-activated normal neutrophils failed to augment adherence of neutrophils from patient 1 (less than 5% adherence).(ABSTRACT TRUNCATED AT 400 WORDS)

Oncolytic HSV and PI3K inhibitor BKM120 synergize to promote killing of prostate cancer stem-like cells.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e16503-e16503
Author(s):  
Lei Wang
Keyword(s):  
Prostate Cancer ◽  
Tumor Model ◽  
Complete Regression ◽  
Novel Therapies ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Anti Tumor Activity

e16503 Background: Novel therapies to override chemo-radiation resistance in prostate cancer (PCa) are needed. Prostate cancer sphere-forming cells (PCSCs) (also termed prostate cancer stem-like cells) likely participate in tumor progression and recurrence and are important therapeutic targets. Methods: We established PCSC-enriched spheres by culturing human (DU145) and murine (TRAMP-C2) PCa cells in growth factor-defined serum-free medium, and characterized stem-like properties of clonogenicity and tumorigenicity in vivo. The efficacy of two different oHSVs (G47∆ and MG18L) in PCSCs was tested alone and in combination with radiation, chemotherapy, and inhibitors of PI3K, Wnt, and NOTCH in vitro, and G47∆ with BKM120 in a PCSC-derived tumor model in vivo. Results: PCSCs were more tumorigenic than serum-cultured parental cells. Human and murine PCSCs were sensitive to oHSV and BKM120 killing in vitro, while the combination was synergistic. In contrast, oHSV combined with radiation, docetaxel, Wnt, or NOTCH inhibitors was not. In athymic mice bearing DU145 PCSC-derived tumors, combination intra-tumoral G47∆ and systemic BKM120 induced complete regression of tumors in 2 of 7 animals, and exhibited superior anti-tumor activity compared to either monotherapy alone, with no detectable toxicity. Conclusions: oHSV synergizes with BKM120 in killing PCSCs in vitro and the combination markedly inhibits tumor growth even inducing regression in vivo.

scholarly journals The role of neutrophil membrane glycoprotein GP-150 in neutrophil adherence to endothelium in vitro

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 167-178
Author(s):  
JM Harlan ◽  
PD Killen ◽  
FM Senecal ◽  
BR Schwartz ◽  
EK Yee ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Membrane Glycoprotein ◽  
Ionophore A23187 ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Endothelial Monolayers ◽  
Neutrophil Adherence

We have previously described two patients with a congenital defect in neutrophil function characterized by an inability to form pus. The patients' neutrophils lack a membrane glycoprotein of mol wt 150,000 daltons (GP-150) on analysis by SDS-PAGE. This glycoprotein is part of a membrane antigen complex recognized by the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produces defects in chemotaxis and phagocytosis in vitro similar to those observed in the patients. Since neutrophil adherence to vascular endothelium is prerequisite to neutrophil emigration in vivo, we examined the interaction of the patients' neutrophils and normal neutrophils treated with MoAb 60.3 with cultured endothelium. Adherence was determined as the percentage of 51Cr-labeled purified peripheral blood neutrophils which remained adherent to plastic wells or endothelial monolayers after a 45-minute incubation at 37 degrees C. The percentage of neutrophils from patient 1 remaining adherent to uncoated, fibronectin-coated, or laminin-coated plastic was similar to that observed in normal neutrophils (55% to 84% adherence with normal neutrophils v 73% to 78% adherence with the patient's neutrophils and 63% to 82% adherence with MoAb 60.3-treated normal neutrophils). The adherence of the neutrophils from patient 1 and MoAb 60.3-treated normal neutrophils to human or bovine endothelium in serum-free medium was also not significantly different from that observed in normal neutrophils (less than 10% adherence with normal, MoAb 60.3-treated, and patient neutrophils). In medium containing 10% autologous or heterologous human plasma, however, the adherence of neutrophils from patient 1 or MoAb 60.3-treated normal neutrophils to endothelial monolayers was significantly reduced (35% +/- 7% of normal neutrophils in seven experiments). Although phorbol myristate acetate (PMA) (10 ng/mL) and calcium ionophore A23187 (10(-5) mol/L) markedly increased the adherence of normal neutrophils to endothelial monolayers in serum- free medium (40% to 85% adherence), neither agent increased the adherence of the neutrophils from patient 1 or normal neutrophils treated with MoAb 60.3 (less than 5% adherence). The adherence of PMA- activated neutrophils from patient 2 to endothelial monolayers was also markedly decreased when compared with that of normal neutrophils. Postsecretory cell-free supernatants from PMA-activated normal neutrophils failed to augment adherence of neutrophils from patient 1 (less than 5% adherence).(ABSTRACT TRUNCATED AT 400 WORDS)

Erythrocyte survival is promoted by plasma and suppressed by a Bak-derived BH3 peptide that interacts with membrane-associated Bcl-XL

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3439-3448 ◽  
Author(s):  
Melanie Walsh ◽  
Robert J. Lutz ◽  
Thomas G. Cotter ◽  
Rosemary O'Connor
Keyword(s):  
Cell Death ◽  
Intracellular Calcium ◽  
Culture System ◽  
Caspase Inhibitor ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Erythrocyte Survival

Abstract Erythrocytes have a defined lifespan in vivo, and the signals that maintain their survival in circulation or trigger their death are unknown. Here, we investigated the control of erythrocyte survival and death in an in vitro culture system where erythrocytes survived for 10 days in serum-free medium in the presence or absence of bovine serum. Death of the cells in culture was correlated with increased exposure of phosphatidylserine and increased levels of intracellular calcium. Cell death could be suppressed by supplementing the medium with human plasma or serum, resulting in a doubling of the lifespan to 20 days. Freshly isolated erythrocytes and cultured erythrocytes were both found to express Bcl-XL and, to a lesser extent, Bak in membrane protein extracts. Treatment of the cells with a Bak-derived BH3 peptide fused to the internalization sequence of the antennapedia protein, which has previously been shown to enter cells by diffusion and antagonize Bcl-XL, resulted in substantial cell death in erythrocyte cultures. BH3-induced death was accompanied by an immediate increase in accumulation of intracellular calcium and could be suppressed by plasma, but not by the caspase inhibitor zVAD. A BH3 peptide mutated at amino acid 78 of full-length Bak required for heterodimerization with Bcl-XL had no effect on cell viability or calcium levels. We conclude that the BH3 peptide accelerates erythrocyte death through antagonization of Bcl-XL. The data suggest that erythrocyte survival is promoted by survival factors in plasma and by membrane-associated Bcl-XL.

In vitro studies on the control of trophoblast outgrowth in the mouse

Development ◽  
1973 ◽  
Vol 30 (1) ◽  
pp. 21-30
Author(s):  
E. J. Jenkinson ◽  
I. B. Wilson
Keyword(s):  
Blastocyst Stage ◽  
Mouse Embryos ◽  
In Vitro Studies ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Uterine Environment ◽  
Adhesive Interactions

When placed in serum-free medium on reconstituted collagen surfaces re-implantation mouse embryos are capable of producing characteristic trophoblast outgrowths. Previously this pattern of differentiation has been considered to be essentially dependent on the presence of serum macromolecules. Such activity is expressed only at the late blastocyst stage and is qualitatively different from the adhesive interactions between blastomeres earlier in development. The development of the properties responsible for outgrowth is intrinsic to the blastocyst, being independent of stimulation by exposure either to the uterine environment or to whole serum. The significance of these observations related to implantation control in vivo is discussed.

THE INFLUENCE OF SERUM ON THE UPTAKE, CONVERSION AND ACTION OF DIHYDROTESTOSTERONE IN RAT PROSTATE GLANDS IN ORGAN CULTURE

1975 ◽  
Vol 64 (2) ◽  
pp. 289-297 ◽  
Author(s):  
ILSE LASNITZKI ◽  
HILARY R. FRANKLIN
Keyword(s):  
Organ Culture ◽  
Horse Serum ◽  
Target Tissue ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Human Pregnancy ◽  
Human Male ◽  
Columnar Cells

SUMMARY The influence of serum on the uptake, conversion and action of dihydrotestosterone in relation to the sex steroid binding protein, TeBG, has been investigated in rat ventral prostates in organ culture. The organs were incubated with [1,2-3H]dihydrotestosterone in: (1) serum-free medium, (2) horse serum, foetal and newborn bovine serum or (3) human male and human pregnancy serum. With all sera the uptake of dihydrotestosterone fell with rising serum concentration, at first steeply and then more gradually. At the same concentration, the uptake was significantly lower in explants incubated with human pregnancy serum than in those kept with human male serum. The conversion of dihydrotestosterone to androstanediol followed the same pattern and less androstanediol was formed in the presence of pregnancy serum. Since pregnancy serum contains higher amounts of TeBG than male serum, the lowered uptake suggests that only the free hormone was available to the target organ. Addition of unlabelled dihydrotestosterone resulted in a higher uptake than that measured in explants incubated with the labelled steroid only. The effect of the human sera on uptake and conversion was correlated with the androgenic activity of dihydrotestosterone applied at physiological concentrations and expressed as the percentage of secretory columnar cells present. The degree of maintenance closely corresponded to the uptake of the hormone. In serum-free medium, the number of columnar cells approached the values found in vivo, with male serum their number, though reduced, was still substantial, with pregnancy serum it was extremely low. It is concluded that the amounts of TeBG present in serum regulate the supply of the hormone to the target tissue and thus control its biological action.

scholarly journals Quantification of Internalized Silica Nanoparticles via STED Microscopy

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Henrike Peuschel ◽  
Thomas Ruckelshausen ◽  
Christian Cavelius ◽  
Annette Kraegeloh
Keyword(s):  
Silica Nanoparticles ◽  
Super Resolution ◽  
Engineered Nanoparticles ◽  
Stimulated Emission ◽  
Alveolar Type ◽  
Serum Free Medium ◽  
Free Medium ◽  
Essential Information ◽  
Serum Free

The development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2×1010particles mL−1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to thein vitrosedimentation, diffusion, and dosimetry (ISDD) model 20–27% of the particles sedimented. In comparison, 102-103NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 1012particles mL−1) of the smaller particles induced cytotoxicity.

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