Shear sensitivity of three hybridoma cell lines in suspension culture

1987 ◽  
pp. 316-327 ◽  
Author(s):  
C.G. Smith ◽  
P.F. Greenfield ◽  
D.H. Randerson
Keyword(s):  
Suspension Culture ◽  
Cell Lines ◽  
Shear Sensitivity ◽  
Hybridoma Cell Lines

scholarly journals Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis

2006 ◽  
Vol 11 (8) ◽  
pp. 922-932 ◽  
Author(s):  
Andrea Ivascu ◽  
Manfred Kubbies
Keyword(s):  
Tumor Cell ◽  
Suspension Culture ◽  
Cell Lines ◽  
Cell Function ◽  
Tumor Cell Lines ◽  
Proliferating Cells ◽  
Basement Membrane Extract ◽  
Rapid Generation ◽  
Size Heterogeneity

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

scholarly journals Factors Affecting the Selection of Callus Cell Lines and the Preparation of the Cell Suspension Culture of Artemisia annua L.

2014 ◽  
Vol 23 (2) ◽  
pp. 157-163 ◽  
Author(s):  
Ch’ng Song Jin ◽  
Chan Lai Keng
Keyword(s):  
Suspension Culture ◽  
Cell Suspension ◽  
Cell Lines ◽  
Cell Suspension Culture ◽  
Artemisia Annua ◽  
Leaf Explants ◽  
In Vitro Production ◽  
Callus Cell ◽  
Alternative Mode ◽  
Artemisia Annua L

Artemisinin, an important antimalarial drug against multidrug resistant strains of Plasmodium, can be produced in Artemisia annua L. Field production of artemisinin is affected by environmental condition and geographical location. In vitro production via cell suspension culture is an alternative mode and cell line selection is important to ensure sustainable production of biomass and artemisinin. Callus cell lines were derived from the leaf explants of five A. annua clones grown in two different locations in Vietnam. Thirty-four callus cell lines with consistent growth index (GI) were selected from these five clones and were categorized into fast (GI > 20), intermediate (GI 15 - 20) and slow (GI < 15) growing groups. The selected lines were found to have different morphology in term of colour and texture. The callus texture did affect the cell growth of A. annua in which the friable callus type showed faster, consistent and sustainable cell biomass production.D. O. I.http://dx.doi.org/10.3329/ptcb.v23i2.17507Plant Tissue Cult. & Biotech. 23(2): 157-163, 2013  (December)

scholarly journals Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament in vivo

2016 ◽  
Author(s):  
Maureen M. Mundia ◽  
Alissa C. Magwood ◽  
Mark D. Baker
Keyword(s):  
Homologous Recombination ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Atp Hydrolysis ◽  
Dominant Negative ◽  
Strand Exchange ◽  
Wild Type ◽  
Hybridoma Cell Lines ◽  
Insight Into

ABSTRACTIn this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3’ polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3’ polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3’ polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3’ polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

scholarly journals LvsA, a Protein Related to the Mouse Beige Protein, Is Required for Cytokinesis in Dictyostelium

1999 ◽  
Vol 10 (12) ◽  
pp. 4429-4439 ◽  
Author(s):  
Eunice Kwak ◽  
Noel Gerald ◽  
Denis A. Larochelle ◽  
Kalpa K. Vithalani ◽  
Maria L. Niswonger ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Suspension Culture ◽  
Cell Lines ◽  
Heavy Chain ◽  
Large Volume ◽  
Myosin Ii ◽  
Cleavage Furrow ◽  
Membrane Processing ◽  
Chediak Higashi Syndrome ◽  
Unusual Phenotype

We isolated a Dictyostelium cytokinesis mutant with a defect in a novel locus called large volume sphere A (lvsA). lvsA mutants exhibit an unusual phenotype when attempting to undergo cytokinesis in suspension culture. Early in cytokinesis, they initiate furrow formation with concomitant myosin II localization at the cleavage furrow. However, the furrow is later disrupted by a bulge that forms in the middle of the cell. This bulge is bounded by furrows on both sides, which are often enriched in myosin II. The bulge can increase and decrease in size multiple times as the cell attempts to divide. Interestingly, this phenotype is similar to the cytokinesis failure of Dictyosteliumclathrin heavy-chain mutants. Furthermore, both cell lines cap ConA receptors but form only a C-shaped loose cap. Unlike clathrin mutants,lvsA mutants are not defective in endocytosis or development. The LvsA protein shares several domains in common with the molecules beige and Chediak–Higashi syndrome proteins that are important for lysosomal membrane traffic. Thus, on the basis of the sequence analysis of the LvsA protein and the phenotype of thelvsA mutants, we postulate that LvsA plays an important role in a membrane-processing pathway that is essential for cytokinesis.

Three Highly Sensitive and High-Throughput Serological Approaches for Detecting Dickeya dadantii in Sweet Potato

Plant Disease ◽  
2021 ◽  
Vol 105 (4) ◽  
pp. 832-839
Author(s):  
Wanqin He ◽  
Deqing Huang ◽  
Jiayu Wu ◽  
Xue Li ◽  
Yajuan Qian ◽  
...  
Keyword(s):  
Sweet Potato ◽  
Cell Lines ◽  
Immunosorbent Assay ◽  
Root Rot ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dickeya Dadantii ◽  
Crude Extracts ◽  
Tissue Print ◽  
Hybridoma Cell Lines ◽  
Dot Elisa

Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection methods is crucial for diagnosis and management of this disease in field. Here, we report the production of four hybridoma cell lines (25C4, 16C10, 9B1, and 9H10) using Dickeya dadantii strain FY1710 as an immunogen. Monoclonal antibodies (MAbs) produced by these four hybridoma cell lines were highly specific and sensitive for D. dadantii detection. Indirect enzyme-linked immunosorbent assay (indirect-ELISA) results showed that the four MAbs 25C4, 16C10, 9B1, and 9H10 could detect D. dadantii in suspensions diluted to 4.89 × 104, 4.89 × 104, 9.78 × 104, and 9.78 × 104 CFU/ml, respectively. Furthermore, all four MAbs can react strongly and specifically with all four D. dadantii strains used in this study, not with the other seven tested bacterial strains. Using these four MAbs, three different serological approaches, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-ELISA, and tissue-print-ELISA, were developed for detection of D. dadantii in crude extracts prepared from field-collected sweet potato plants. Among these three methods, TAS-ELISA and dot-ELISA were used to detect D. dadantii in suspensions diluted up to 1.23 × 104 and 1.17 × 106 CFU/ml, respectively, or in sweet potato crude extracts diluted up to 1:3,840 and 1:1,920 (wt/vol, grams per milliliter), respectively. Surprisingly, both TAS-ELISA and dot-ELISA serological approaches were more sensitive than the conventional PCR. Analyses using field-collected sweet potato samples showed that the newly developed TAS-ELISA, dot-ELISA, or tissue-print-ELISA were reliable in detecting D. dadantii in sweet potato tissues. Thus, the three serological approaches were highly valuable for diagnosis of stem and root rot in sweet potato production.

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