Computational protocol: Poly-γ-glutamic acid/chitosan nanogel greatly enhances the efficacy and heterosubtypic cross-reactivity of H1N1 pandemic influenza vaccine

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Protocol publication

[…] To examine antigen uptake and processing, 1 μg/mL DQ-OVA (Molecular Probes), which is OVA conjugated to a self-quenched dye that emits green fluorescence upon degradation, was mixed with 400 μg/mL of an adjuvant and incubated for 16 h at 4 °C. DCs were plated onto μ-Slides (Ibidi GmbH) and stimulated with the mixture for 4 h at 37 °C followed by incubating with LysoTracker and DAPI (Molecular Probes). To examine the accumulation of MHC-I in acidic compartments, DCs were incubated with a mixture of OVA-Texas red (1 μg/mL; Molecular Probes) with either alum or PC nanogel for 3 h at 37 °C and then exposed to LysoTracker for the last 30 min. The cells were fixed, permeabilized and stained with an anti-MHC-I (BD) followed by Alexa488-conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were counterstained with DAPI. The stained cells were examined with an LSM5 Pascal confocal fluorescence microscope (Carl Zeiss). The percentages of cells in which MHC-I colocalized with LysoTracker or OVA (Manders’ coefficient) in the insets were calculated using the JACoP plugin of the ImageJ software. […]

Pipeline specifications

Software tools JACoP, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Human poliovirus 1 Mahoney, Mus musculus
Diseases Influenza, Human, HIV Infections