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. 2019 May 13;10:2144. doi: 10.1038/s41467-019-09879-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — INSPECT-3D workflow, experimental system and population analysis of pathogen spread in 3D collagen. a Schematic workflow depicting the different steps of INSPECT-3D. Step 1: Establishment of a 3D culture system (here: primary human CD4 T lymphocytes in collagen to study HIV-1). Steps 2 and 3: Generation of quantitative and kinetic data on pathogen spread on the population level (step 2) and on the level of single pathogens (step 3). Step 4: Use quantitative and time-resolved information from steps 2 and 3 to parametrize a model based on ordinary differential equations (ODE) describing the kinetics of pathogen spread. Step 5: Generation of quantitative and kinetic data on cell motility and contact parameters. Step 6: Use quantitative and time-resolved information from step 5 to generate a cellular Potts model describing cell motility. Step 7: Combine both modeling approaches (steps 4 and 7) into an in silico integrated spatial infection model. Step 8: Experimental validation of predictions from the in silico integrated spatial infection model. Infected cells: green, target cells: red, virus: green asterisks, CD8 cells: purple, newly infected cells in eclipse phase: orange. b Schematic overview of experimental procedure of PBMC isolation, activation, infection and set up of long-term infection cultures or short-term imaging. Infected T cells (green) can be separated from uninfected T cells (red) by MACS sorting of cells infected with the HIV-1 variant NL4.3Disp.YFP. c Schematic overview of the parameters that can be quantified by INSPECT-3D. Depicted in the top panel are schematic views of 2D suspension and 3D collagen cultures with uninfected and infected cells in red and green, respectively. Asterisks: virus. The middle panel shows a wide-field micrograph of cell density and arrangement, the lower panel a still image from a movie of a 3D culture with uninfected (red, PKH26 stained) and infected (green, PKH67 stained) cells. Scale bars: 40 µm. d–f HIV-1 spread in suspension or collagen. d Virus concentration in supernatants determined by SG-PERT. e Percentage of infected (p24+) CD4 T cells determined by flow cytometry. f T cell depletion expressed as residual CD4 T cells relative to the respective T20 control, which was set to 100% (dashed line). Mean and standard deviation from parallel triplicate infections of cells from the same donors are shown