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Multiparameter Flow Cytometry Best Practices

Multiparameter flow cytometry is an important tool for biomedical scientists, especially for preclinical drug and vaccine research. It is an invaluable tool for detailed analysis of receptors, signaling, and effector molecules as well as nucleic acids in individual cells and cell populations. Technological advancements have made it possible to design and execute flow cytometry studies that simultaneously evaluate multiple parameters. However, evaluating multiple parameters at the cellular level is not an easy task.  Below are some sample preparation, panel design, and data analysis aspects for new and inexperienced flow cytometry users to consider.

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Biotinylation of Antibodies and Antigens

Antibodies bind specifically to the antigens that induce their production. This specificity is an advantage when identifying and quantifying proteins in an unknown sample mixture. This is the basis for immunoassays such as Western blots and enzyme-linked immunosorbent assays (ELISA).  In some cases, structural similarities between proteins and interference from diluent ingredients can lead to nonspecific binding resulting in high background and false-positive signals. This is a very common problem with polyclonal antibodies and in multiplexed assays.

Biotin and streptavidin offer solutions for improving specificity and reducing background without disrupting the normal biological function of the proteins.   Biotin (Vitamin B7) is a water-soluble vitamin that covalently binds to and stabilizes proteins in a biological system. Due to its small molecular weight and simple chemical structure biotin conjugation does not affect the biological function of bound proteins.

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Closing the gate on SARS-CoV-2: A VSV pseudotype neutralization assay targeting the key to viral entry

To address the urgent and immediate need for R&D tools for the fight against the public health threat from SARS-CoV-2, IBT Bioservices now offers a pseudotype virus system to assay inhibition of infectivity in a BSL-2 environment. Briefly, Vesicular Stomatitis virus (VSV) glycoprotein gene (G) has been substituted with SARS-CoV-2 Spike Protein (rVSV pseudotyped SARS-CoV-2 Spike). The recombinant rVSV-DG SARS-CoV-2 Spike has been incorporated into a convenient luciferase-based neutralization assay for evaluating the efficacy of drug candidates that target Spike-mediated infection (Figure 1). This system is similar to previously published and validated VSV pseudotype platform for Ebolavirus and Marburgvirus1,2,3.

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Staphylococcus aureus skin infection model

The increasing rate of bacterial resistance creates a challenging environment for the development of therapies for bacterial infections. Bacterial skin infections are one of the leading manifestations of infectious diseases in the world. Staphylococcus aureus—both Methicillin-resistant (MRSA) and Methicillin-Sensitive (MSSA) strains—is the leading cause of skin and soft tissue infections in the USA. It is the leading cause of hospital-associated (HA) and community-associated (CA) infections worldwide.

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Coronavirus: The Origin Story

In recent years, animal-to-human crossovers have been observed with Nipah virus in Malaysia, and Ebola and Marburg viruses in Africa. SARS-CoV-2 is just one among three 21st century animal-to-human Coronavirus spillover events. Considering the high rate of mutation among RNA viruses, the number of animal coronaviruses, and the mixing of animals into densely populated areas spillover is not unexpected.

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Flow Cytometry: A Vital Tool for Evaluating Vaccine Effectiveness

Flow cytometry uses light scattering, light excitation and emission of fluorochrome molecules to gather detailed information about specific cells or particles of interest. Using antibody-fluorophore conjugates flow cytometry allows researchers to draw phenotype-dependent conclusions about cellular phenotype and function.  The fluorophores present on bound antibodies excited by lasers yield detailed…

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