Journal: BMC Biotechnology

Article Title: Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF

doi: 10.1186/s12896-024-00853-6

Figure Lengend Snippet: Overview of experiments assessing the influence of buffer formulation on the thermal stability and infectivity of AAV6.2FF. A In vitro assay design to evaluate the effect of buffer formulation on the infectivity and stability of AAV6.2FF-ffLuc at different temperatures and upon exposure to multiple rounds of freeze-thaw. AAV6.2FF-ffLuc vector was formulated in PBS or 0.001% PF-68-PBS and aliquoted in 1x10 9 vg doses into 1.5 mL microfuge tubes. Samples ( n =4) were subjected to incubation at -20℃, 4℃, 21℃, 37℃, and 55℃ for durations of 30 minutes (m), 1, 4, 12 or 24 hours (h) to 2, 3, or 5 days (d) or 1 or 2 weeks (w). Another set of samples ( n =4) were subjected to 1 to 10 freeze-thaw cycles. All samples were evaluated for transducing activity in HEK293 cells ( n =4) or for particle integrity by qPCR ( n =3). B In vivo assay to investigate the effects of buffer formulation on the in vivo transducing properties of AAV6.2FF-eGFP. C57BL/6 mice ( n =7) were intranasally administered 1x10 11 vg AAV6.2FF-eGFP with or without 0.001% PF-68. Mice were euthanized 5 weeks post vector administration and lungs were harvested, dissociated, stained for CD45, EpCAM, and 7AAD and subjected to flow cytometric analysis

Article Snippet: Lysates were analyzed for luciferase expression using the Pierce™ Firefly Luciferase Glow Assay Kit (ThermoFisher, cat. no. 16176) and the GloMax®-Multi Detection System plate reader (Promega) as per the manufacturer’s instructions

Techniques: Formulation, Infection, In Vitro, Plasmid Preparation, Incubation, Activity Assay, In Vivo, Staining

Journal: BMC Biotechnology

Article Title: Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF

doi: 10.1186/s12896-024-00853-6

Figure Lengend Snippet: The effect of temperature on AAV6.2FF infectivity in different buffer formulations. Aliquots of 1x10 9 vg of AAV6.2FF-ffLuc formulated PBS or PBS supplemented with 0.001% PF-68 were exposed to a range of temperatures and durations. Following treatment, vector aliquots were applied to HEK293 cells and luciferase expression measured in relative luminescent units (RLU) 72 hours later. The temperatures tested were as follows: A -20℃, B 4℃, C 21℃, D 37℃, or E 55℃. A two-way ANOVA test was performed between the two buffer formulations in all exposure and treatment conditions. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001

Article Snippet: Lysates were analyzed for luciferase expression using the Pierce™ Firefly Luciferase Glow Assay Kit (ThermoFisher, cat. no. 16176) and the GloMax®-Multi Detection System plate reader (Promega) as per the manufacturer’s instructions

Techniques: Infection, Plasmid Preparation, Luciferase, Expressing

Journal: BMC Biotechnology

Article Title: Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF

doi: 10.1186/s12896-024-00853-6

Figure Lengend Snippet: The effect of temperature on AAV6.2FF particle stability in different buffer formulations. Aliquots of 1x10 9 vg of AAV6.2FF-ffLuc formulated PBS or PBS supplemented with 0.001% PF-68 were exposed to a range of temperatures and durations. Following treatment, viral DNA was extracted and quantified by qPCR. The conditions tested were as follows: A -20℃, B 4℃, C 21℃, D 37℃, or E 55℃. A two-way ANOVA test was performed between the two buffer formulations in all exposure and treatment conditions. ns (not significant), * P ≤ 0.05, and **** P ≤ 0.0001

Article Snippet: Lysates were analyzed for luciferase expression using the Pierce™ Firefly Luciferase Glow Assay Kit (ThermoFisher, cat. no. 16176) and the GloMax®-Multi Detection System plate reader (Promega) as per the manufacturer’s instructions

Techniques: