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. 2020 Feb 12;12(2):206. doi: 10.3390/v12020206

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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BlaM HIV-1 fusion calibration utilizing intensity-based FRET approaches and FRET-FLIM. (A) Images of the BlaM assay in TZM-bl cells exposed to different MOIs (1, 2, 5 and 10) of HIV-1_JFRL virions, analyzed using FRET intensity ratio and the fraction of interacting donor (f_D). Images are pseudocolored in red (fusion negative) and green (fusion positive). Scale bars are 20 µm. The statistics coming from the two methods, (B) FRET intensity and (C) f_D, are presented in which every dot represents a single cell (n > 50 cells per condition from one experiment). The green dotted line represents the threshold taken from TZM-bl cells exposed to HIV-1 without spikes (No Env) virions. (D,E) The calibration curves for both methods are presented together with the linear regression and equation/s. The calibration curves were obtained from the data points of three independent experiment (Exp. 1–3). Each symbol represents the mean and SD per condition, per experiment.