Transfection of insect cell in suspension for efficient baculovirus generation

Baculovirus (BV) mediated insect cell expression system utilizes transfection as a first step to introduce recombinant baculovirus DNA into insect cells. Many labs are still relying on the conventional liposome based transfection method in adherent culture. Here we describe a more efficient method that can replace the existing method. The beauty of this method is minimal intermediate manipulation of culture during transfection and virus generation. This significantly reduces the chances of cross contamination of viruses while handling multiple targets and constructs as well as the other microbial contamination. The method is economical and doesn’t require any special adjustment in existing labs

Affordable uniform isotope labeling with 2H, 13C and 15N in insect cells.

A simple and affordable protocol was developed to produce uni-form labeled proteins in insect cells. Incorporation levels of 80% can be achieved for 15N and 13C and yields are comparable to expression in full media. For 2H,15N and 2H,13C,15N labeling, incorporation is slightly lower with 75% and 73%, respectively, and yields are typically two-fold reduced. The media were optimized for simplicity, reproducibility, isotope incorporation and cost. They consist of only five components, which are all commercially available at less than 8% of the costs than labeling media from vendors. High isotope incorporation and low cost are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The approach was applied to several cytosolic and secreted target proteins

Insect cell culture in reagent bottles

Growing insect cells with high air space in culture vessel is common from the early development of suspension cell culture. We believed and followed it with the hope that it allows sufficient air for optimal cell growth. However, we missed to identify how much air exactly cells need for its growth and multiplication. Here we present the innovative method that completely changed the way we run insect cell culture. The method is easy to adapt, cost-effective and useful for both academic and industrial research labs. We believe this method will revolutionize the way we run insect cell culture by increasing throughput in a cost-effective way

Uniform isotope labeling of proteins expressed in insect cells using a custom medium

Production of proteins uniformly labeled with 2H, 13C and 15N isotopes is essential for advanced NMR studies. Here we present an affordable strategy for uniform isotope labeling in the insect cell system. Compared to commercial media the cost of the newly developed media are about 10-fold lower at comparable isotope incorporation. Additionally, there is no restriction on the labeling pattern (only 15N and 13C,15N are available commercially), but 2H, 13C and 15N in every combination can be labeled. The method was evaluated by the NMR studies on expressed proteins with four different uniform labeling patterns

Insect cell culture in reagent bottles

Growing insect cells with high air space in culture vessel is common from the early development of suspension cell culture. We believed and followed it with the hope that it allows sufficient air for optimal cell growth. However, we missed to identify how much air exactly cells need for its growth and multiplication. Here we present the innovative method that changed the way we run insect cell culture. The method is easy to adapt, cost-effective and useful for both academic and industrial research labs. We believe this method will revolutionize the way we run insect cell culture by increasing throughput in a cost-effective way

Somatic/affective symptoms, but not cognitive/affective symptoms, of depression after acute coronary syndrome are associated with 12-month all-cause mortality

Background: Symptom dimensions of post myocardial infarction (MI) depression may be differently related to prognosis. Somatic/affective symptoms appear to be associated with a worse cardiac outcome than cognitive/affective symptoms. We examined the relationship between depressive symptom dimensions following acute coronary syndrome (ACS) and both disease severity and all-cause mortality. Methods: Patients (n=913) who had unstable angina pectoris or MI were recruited from 12 coronary care units between 1997 and 1999. Measurements included sociodemographic and clinical data and the Beck Depression Inventory (BDI). Endpoint was all-cause mortality at 12-month follow-up. Results: Principal component analysis revealed two components, somatic/affective and cognitive/affective symptoms of depression. Somatic/affective symptoms of depression (odds ratio (OR): 1.49; 95% confidence interval (CI): 1.23-1.81; p Limitations: Time to death was not available. Conclusions: This study showed that only somatic/affective depressive symptoms were associated with disease severity and all-cause mortality in ACS patients. More research is needed to evaluate the differential associations of somatic/affective and cognitive/affective depressive symptoms with cardiac outcomes and the underlying mechanisms. (C) 2010 Elsevier B.V. All rights reserved

An Organotypic Human Lymph Node Model Reveals the Importance of Fibroblastic Reticular Cells for Dendritic Cell Function

BACKGROUND: Human lymph node (HuLN) models have emerged with invaluable potential for immunological research and therapeutic application given their fundamental role in human health and disease. While fibroblastic reticular cells (FRCs) are instrumental to HuLN functioning, their inclusion and recognition of importance for organotypic in vitro lymphoid models remain limited.METHODS: Here, we established an in vitro three-dimensional (3D) model in a collagen-fibrin hydrogel with primary FRCs and a dendritic cell (DC) cell line (MUTZ-3 DC). To study and characterise the cellular interactions seen in this 3D FRC-DC organotypic model compared to the native HuLN; flow cytometry, immunohistochemistry, immunofluorescence and cytokine/chemokine analysis were performed.RESULTS: FRCs were pivotal for survival, proliferation and localisation of MUTZ-3 DCs. Additionally, we found that CD1a expression was absent on MUTZ-3 DCs that developed in the presence of FRCs during cytokine-induced MUTZ-3 DC differentiation, which was also seen with primary monocyte-derived DCs (moDCs). This phenotype resembled HuLN-resident DCs, which we detected in primary HuLNs, and these CD1a - MUTZ-3 DCs induced T cell proliferation within a mixed leukocyte reaction (MLR), indicating a functional DC status. FRCs expressed podoplanin (PDPN), CD90 (Thy-1), CD146 (MCAM) and Gremlin-1, thereby resembling the DC supporting stromal cell subset identified in HuLNs. CONCLUSION: This 3D FRC-DC organotypic model highlights the influence and importance of FRCs for DC functioning in a more realistic HuLN microenvironment. As such, this work provides a starting point for the development of an in vitro HuLN.</p