Interaction of Biomphalaria cells in primary cultures with Schistosoma mansoni sporocysts

November 12, 2020
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eaaci2015

Culture of Intestinal Epithelial Cell Monolayers and Their Use in Multiplex Macromolecular Permeability Assays for In Vitro Analysis of Tight Junction Size Selectivity

 

Tight junctions form a selectively permeable barrier that limits paracellular flux across epithelial-lined surfaces. Small molecules (less than ∼8 Å diameter) can traverse the junction via the size- and charge-selective, high-conductance pore pathway. In contrast, the low-conductance leak pathway accommodates larger macromolecules (up to ∼100 Å diameter) and is not charge-selective. Flux across the tight junction-independent, high-conductance, non-selective, unrestricted pathway occurs at sites of epithelial damage.

 

Cytokines can regulate each of these pathways, but commonly used measures of barrier function cannot discriminate between tight junction regulation and epithelial damage. This article describes methods for culturing intestinal epithelial cell monolayers and assessing the impact of cytokine treatment on leak and unrestricted pathway permeabilities. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation and culture of cell monolayers in Transwells Basic Protocol 2: Assessment of cytokine (IFNγ and TNF) treatment effects on barrier function Support Protocol: Immunofluorescent staining of monolayers Basic Protocol 3: Multiplex flux assay.

 

 

Interaction of Biomphalaria cells in primary cultures with Schistosoma mansoni sporocysts

Introduction: Biomphalaria snails may display varying levels of susceptibility to Schistosoma mansoni infection. We have been developing an in vitro model to study the interaction between the snail and the parasite, using tissue-derived cell cultures from Biomphalaria.
Methods: The digestive gland- and kidney-derived cells from primary cultures of resistant (B. tenagophila Taim) and susceptible (B. tenagophila HM and B. glabrata BH) strains of Biomphalaria were exposed to S. mansoni sporocysts.
Results: S. mansoni sporocysts were surrounded and encapsulated exclusively by cells derived from the digestive gland (DG) of B. tenagophila Taim. The process was followed by a marked decrease in the number of free sporocysts in the culture medium. The morphological characteristics of DG-derived cells in culture have been described.
Conclusions: Cells derived from DG (but not SK) primary cultures of B. tenagophila Taim may participate in S. mansoni sporocyst control.
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eaaci2015

myo-Inositol
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myo-Inositol
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myo-Inositol
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myo-Inositol
GC4902-500G 500 g
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myo-Inositol
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myo-Inositol
B7903-500 500 mg
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Myo-inositol
TBZ2449 20mg Ask for price

Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *Medium Removal*
36330 1 Plate
EUR 132

Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *Medium Removal*
36331 10 Plates
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Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *Medium Removal*
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Advanced Medium
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M9 Medium
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NeuroProgenitor Medium
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Inositol 2-dehydrogenase/D-chiro-inositol 3-dehydrogenase Polyclonal Antibody
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Inositol Monophosphatase 1 (Recombinant)
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Comparison of VEGF-A secretion from tumor cells under cellular stresses in conventional monolayer culture and microfluidic three-dimensional spheroid models

 

Vascular endothelial growth factor (VEGF) is a major cytokine in tumor biology affecting tumor survival, aggressiveness and pro-angiogenetic activities. In addition, cellular stresses often result in aggressive pro-angiogenetic behavior in tumors. For in vitro study, conventional monolayer cell culture has been broadly exploited; however, it often provides limited information due to its different microenvironment from that in vivo. Recently, three-dimensional (3D) cell spheroid culture provides in vivo-like microenvironments to study tumor biology and their survival mechanisms with better predictive power.
In this work, vascular endothelial growth factor of type A (VEGF-A) secretion from osteosarcoma (MG-63) cells cultured using monolayer and 3D spheroid models under two stress conditions: nutrient deficiency (reduced serum culture) and hypoxia-inducible factor (HIF) inhibition (HIF inhibitor, YC-1) are characterized and systematically compared.
In order to obtain ample sample size for consistent characterization of cellular responses from cancer spheroids under the stresses and compare the responses to those from the conventional monolayer model, a microfluidic spheroid formation and culture device is utilized in the experiments.
In the analysis, cell viability is estimated from captured images, and quantification of VEGF-A secreted from the cells is achieved using enzyme-linked immunosorbent assay (ELISA). The experimental results show that the viabilities decrease when the cells face higher stress levels in both monolayer and 3D spheroid culture models; however, the VEGF-A secretion profiles between the cell culture models are different.
The VEGF-A secretion decreases when the cells face higher stress conditions in the monolayer cell culture. In contrast, for the 3D spheroid culture, the VEGF-A concentration decreases for low stress levels but increases while the stress level is high. The VEGF-A regulation in the 3D models mimics in vivo cases of tumor survival and can provide insightful information to investigate tumor angiogenesis in vitro. The approach developed in this paper provides an efficient method to quantitatively and statistically study tumor growth kinetics and stress responses from highly uniform samples and it can also be applied to compare the underlying biomolecular mechanisms in monolayer and 3D spheroid culture models to elucidate the effects of microenvironments on cellular response in cancer research.

Protection of human induced pluripotent stem cells against shear stress in suspension culture by Bingham plastic fluid

 

Suspension culture is an important method used in the industrial preparation of pluripotent stem cells (PSCs), for regenerative therapy and drug screening. Generally, a suspension culture requires agitation to keep PSC aggregates suspended and to promote mass transfer, but agitation also causes cell damage. In this study, we investigated the use of a Bingham plastic fluid, supplemented with a polysaccharide-based polymer, to preserve PSCs from cell damage in suspension culture. Rheometric analysis showed that the culture medium gained yield stress and became a Bingham plastic fluid, after supplementation with the polymer FP003.

 

A growth/death analysis revealed that 2 days of aggregate formation and 2 days of suspension in the Bingham plastic medium improved cell growth and prevented cell death. After the initial aggregation step, whereas strong agitation (120 rpm) of a conventional culture medium resulted in massive cell death, in the Bingham plastic fluid we obtained the same growth as the normal culture with optimal agitation (90 rpm). This indicates that Bingham plastic fluid protected cells from shear stress in suspension culture and could be used to enhance their robustness when developing a large-scale. This article is protected by copyright. All rights reserved.

 

Metabolic profiling of CHO cell cultures at different working volumes and agitation speeds using spin tube reactors

 

Culture systems based on spin tube reactors have been consolidated in the development of manufacturing processes based on Chinese hamster ovary (CHO) cells. Despite their widespread use, there is little information about the consequences of varying operational setting parameters on the culture performance of recombinant CHO cell lines. Here we investigated the effect of varying working volumes and agitation speeds on cell growth, protein production and cell metabolism of two clonally-derived CHO cell lines (expressing an IgG1 and a ‘difficult-to-express’ fusion protein). Interestingly, low culture volumes increased recombinant protein production and decreased cell growth, while high culture volumes had the opposite effect. Altering agitation speeds exacerbated or moderated the differences observed due to culture volume changes.

 

Combining low agitation rates with high culture volumes suppressed growth and recombinant protein production in CHO cells. Meanwhile, high agitation rates narrowed the differences in culture performance between low and high working volumes. These differences were also reflected in cell metabolism, where low culture volumes enhanced oxidative metabolism (linked to a productive phenotype) and high culture volume generated a metabolic profile that was predominately glycolytic (linked to a proliferative phenotype). Our findings indicate that the culture volume influence on metabolism modulates the balance between cell growth and protein production, a key feature that may be useful to adjust CHO cells towards a more productive phenotype.

 

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2-Amino-6-nitrobenzoic acid
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2-Amino-5-Fluorobenzoic Acid
abx188201-100g 100 g
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3-Amino-2-Methylbenzoic Acid
abx188349-1000g 1000 g
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cis-3-Amino-cyclobutanecarboxylic acid
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2-Amino-4-pyridinecarboxylic acid
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3-Amino-5-bromobenzoic acid
GM0393-25G 25 g
EUR 190

11-(Boc-amino)-undecanoic acid
A-3310.0005 5.0g
EUR 151
Description: Sum Formula: C16H31NO4; CAS# [10436-25-6]

11-(Boc-amino)-undecanoic acid
A-3310.0025 25.0g
EUR 515
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GM8338-10G 10 g
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GM8338-50G 50 g
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1-(Fmoc-amino)-cyclopentanecarboxylic acid
B-3570.0005 5.0g
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anti-D Amino Acid Oxidase
YF-PA23561 50 ul
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Description: Mouse polyclonal to D Amino Acid Oxidase

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Synthetic pyrethroid insecticides
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Synthetic Neurotensin (NT)
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Description: Recombinant Human Neurotensin expressed in: E.coli

Synthetic Phytohemagglutinin (PHA)
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Description: Recombinant Pan-species Phytohemagglutinin expressed in: E.coli

Atpenin A5 (Synthetic)
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Atpenin A5 (Synthetic)
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Fructosyl-amino acid oxidase from Microorganism
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L-Amino Acid Assay Kit (Colorimetric)
MET-5054 100 assays
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D-Amino-Acid Oxidase (DAO) Antibody
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EUR 384

D-Amino-Acid Oxidase (DAO) Antibody
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Fmoc-12-Amino-4,7,10-Trioxadodecanoic Acid
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EUR 370

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2-Amino-Isonicotinic Acid Ethyl Ester
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2-Hydroxy-4-Amino Butanoic Acid
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20-abx184074
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Benzoic acid, o-amino-, ethyl ester
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4-Amino-3-phenylbutyric acid HCl
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EUR 90

(S)-(-)-2-Amino-4-pentenoic acid
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EUR 237

Anti-D-amino-acid oxidase antibody
STJ71455 100 µg
EUR 359

Human D Amino Acid Oxidase Antibody
32963-05111 150 ug
EUR 261

Human D-amino-acid oxidase (DAO)
1-CSB-YP006494HU
  • EUR 430.00
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Description: Recombinant Human D-amino-acid oxidase(DAO) expressed in Yeast

Human D-amino-acid oxidase (DAO)
1-CSB-EP006494HU
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  • EUR 560.00
  • EUR 873.00
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Description: Recombinant Human D-amino-acid oxidase(DAO) expressed in E.coli

Rat D-amino-acid oxidase (Dao)
1-CSB-EP006494RA
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Description: Recombinant Rat D-amino-acid oxidase(Dao) expressed in E.coli

Recombinant rat D-amino-acid oxidase
P1766 100ug Ask for price
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4-Amino-piperidine-4-carboxylic acid
F-3605.0001 1.0g
EUR 345
Description: Sum Formula: C6H12N2O2; CAS# [40951-39-1] net

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F-3605.0005 5.0g
EUR 1301
Description: Sum Formula: C6H12N2O2; CAS# [40951-39-1] net

4-Amino-piperidine-4-carboxylic acid
F-3605.0025 25.0g
EUR 5108
Description: Sum Formula: C6H12N2O2; CAS# [40951-39-1] net

Human Amino Acid Metabolism Primer Library
HAMM-I 1 set
EUR 645

D-amino acid oxidase, human recombinant
P1052-10
EUR 164

D-amino acid oxidase, human recombinant
P1052-50
EUR 588

Anti-D Amino Acid Oxidase (2F5)
YF-MA12633 100 ug
EUR 363
Description: Mouse monoclonal to D Amino Acid Oxidase

Branched Chain Amino Acid Assay Kit
55R-1440 100 assays
EUR 740
Description: Assay Kit for detection of Branched Chain Amino Acids in the research laboratory

D-Amino-Acid Oxidase Recombinant Protein
92-027 0.05 mg
EUR 710.75
Description: D-Amino-Acid Oxidase (DAO) belongs to the DAMOX/DASOX family. DAO is a peroxisomal enzyme which founctions as a homodimer to oxidizes D-amino acids to the corresponding imino acids, producing ammonia and hydrogen peroxide. D-amino-acid oxidase regulates the level of the neuromodulator D-serine in the brain, has a high activity towards D-DOPA and contributes to dopamine synthesis. D-amino-acid oxidase could act as a detoxifying agent which removes D-amino acids accumulated during aging. It also acts on a variety of D-amino acids with a preference for those having small hydrophobic side chains followed by those bearing polar, aromatic, and basic groups.

D-Amino Acid Oxidase/ DAMOX (DAO) Antibody
20-abx301170
  • EUR 411.00
  • EUR 1845.00
  • EUR 599.00
  • EUR 182.00
  • EUR 300.00
  • 100 ug
  • 1 mg
  • 200 ug
  • 20 ug
  • 50 ug

Aromatic-L-Amino-Acid Decarboxylase (DDC) Antibody
20-abx302246
  • EUR 411.00
  • EUR 1845.00
  • EUR 599.00
  • EUR 182.00
  • EUR 300.00
  • 100 ug
  • 1 mg
  • 200 ug
  • 20 ug
  • 50 ug

Native Crotalus adamanteus L-Amino Acid Oxidase
NATE-0366 2 mg
EUR 270
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