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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 simultaneous 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 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 Western blots and enzyme linked immunosorbent assays (ELISA). However, 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 very common problem with polyclonal antibodies, and in  multiplexed assays.

Biotin and streptavidin offer solution for improving specificity and reducing background without disrupting the normal biological function of the proteins.   Biotin (Vitamin B7) is a water-soluble vitamin which 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 disease 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 Nipha virus in Malaysia, 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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Fig. 1 Survival after challenge with INFV H1N1 A/Pert/261/2009 (Tamiflu-resistant strain). Inoculum 1xLD90=1.0E+05 PFU/mouse
Survival after challenge with INFV H1N1 A/Pert/261/2009 (Tamiflu-resistant strain) 1.0E+05 PFU/mouse
Survival and weight change in BALB/c mice challenged with INFV A/ Texas/36/91 (H1N1) and treated with antiviral Osletamivir Phosphate (Tamiflu)
Lung viral load and Survival (30 % weight loss cut-off) in BALB/c mice challenged with INFV H3N2 A/HK/1/68.

Alpha (UK) – B. 1.1.7 / 501Y.V1

amino acid mutations: del69–70 HV, del144 Y, N501Y, A570D, D614G, P681H, T761I, S982A, D1118H

Beta (South Africa) – B.1.351

amino acid mutations: K417N, E484K, N501Y, D614G, A701V

Gamma (Brazil) – P.1

amino acid mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I

Epsilon (Ca, USA) B.1.427

amino acid mutations: L452R, D614G

SARS-CoV-2 Parental Strain Wild Type (Wuhan)
SARS-CoV-2 D614G Variant

amino acid mutations: D614G

Epsilon (Ca, USA) B.1.429

amino acid mutations: S13I, W152C, L452R, D614G

SARS-CoV-2 Delta Variant

amino acid mutations: L452R, E484Q