A data-driven approach to establishing cell motility patterns as predictors of macrophage subtypes and their relation to cell morphology

The motility of macrophages in response to microenvironment stimuli is a hallmark of innate immunity, where macrophages play pro-inflammatory or pro-reparatory roles depending on their activation status during wound healing. Cell size and shape have been informative in defining macrophage subtypes. Studies show pro- and anti-inflammatory macrophages exhibit distinct migratory behaviors, in vitro, in 3D, and *in vivo, *but this link has not been rigorously studied. We apply both morphology and motility-based image processing approaches to analyze live cell images consisting of macrophage phenotypes. Macrophage subtypes are differentiated from primary murine bone marrow-derived macrophages using a potent lipopolysaccharide (LPS) or cytokine interleukin-4 (IL-4). We show that morphology is tightly linked to motility, which leads to our hypothesis that motility analysis could be used alone or in conjunction with morphological features for improved prediction of macrophage subtypes. We ..., • Macrophage Differentiation: Naïve BMDMs (M0) were differentiated into M1 or M2 subtypes. For M1 differentiation, 100 ng/ml lipopolysaccharide (LPS) was used. For M2 differentiation, 20 ng/ml recombinant mouse interleukin-4 (IL-4) was employed.
• Culturing and Activation: The macrophages were seeded in 24-well tissue culture-treated plates and activated as detailed in the accompanying manuscript.
• Imaging: Time-lapse contrast images were captured 48 hours post-stimulation using a Carl Zeiss Observer Z1 inverted microscope with a motorized stage and incubation chamber. Imaging was done using the MetaMorph program with a Retiga R6 CCD camera, under 10× or 20× objective lenses.
, , # Data from: A data-driven approach to establishing cell motility patterns as predictors of macrophage subtypes and their relation to cell morphology

#### Description of the data and file structure

**Time-lapse Images of Murine Macrophages**

Overview:

This dataset comprises time-lapse images of murine macrophages cultured in vitro. The images are provided in TIFF format. Bone marrow was isolated from the femur bones of C57/BL6 mice to generate bone marrow-derived macrophages (BMDMs). We utilized a total of 20 mice, creating five separate batches of BMDMs, with each batch derived from a mixture of bone marrow from 3-6 mice. These BMDMs were then differentiated into naïve (M0), M1, or M2 macrophages using specific stimuli and conditions.

### Files and variables

#### Dataset Details:

• Data Collection Period: September 2019 to May 2021.

• File Naming Convention: Each file is named according to the Macrophage Type, Time Post-Differentiation, Time Interval, Magnification, and Scale.
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