3-dimensional super-resolution microscopy volumes of human PBMCs recorded on a Zeiss LSM980 Airyscan2 laser scanning confocal microscope. Sample preparation and image capture: Live PBMCs were stained with 60 nM MitoTracker Red-CMX-Ros (ThermoFisher, Waltham, MA) either in 6-well plates or in the microgravity chambers for the last 2 hr of the microgravity simulation. At the end of the microgravity simulation cells were immediately fixed by 1:1 mixing the cell suspensions with 2× concentrated fixative (10% Sucrose (w/v) 120 mM KCl, 1% (w/v) glutaraldehyde, 8% (w/v) PFA pH 7.4) and incubated for 15 minutes at room temperature followed by 15 minutes on ice. Fixed cells were washed and stored in PBS until further staining for up to a week at 4 °C. 1 million fixed cells were resuspended in 1 mL of permeabilization solution (0.1% TritonX-100 in PBS) for 5 minutes. After twice washing in PBS, pellets were resuspended in 0.5 mL 1% BSA PBS containing Phalloidin-iFluor-488 (cat# ab176753, Abcam plc., Cambridge, UK) at the manufacturer’s recommended dilution, and were incubated for 90 minutes with gentle agitation. After washing in PBS, cells were stained with Hoechst 33342 (1 µg/mL in PBS) for 10 minutes. The fixed-stained cells were immobilized at 3 × 105 cells per well density in glass-bottom 96-well microplates (Greiner Bio-One, Monroe, NC), which were pre-coated with polyethyleneimine (1:15,000 (w/v)) for 16 hours in a 37 °C incubator, and washed twice with PBS. Microplates with the cell suspensions were centrifuged in a swing plate rotor centrifuge (Eppendorf 5810 R) at 400 × g and for 10 min and then fixed on the surface by adding an equal volume of 8% (w/v) PFA for 5 min. Finally, the fixative was replaced with 100 µL of antifade reagent (Vector Prolong Gold (ThermoFisher)). Samples were imaged immediately after this procedure on a Zeiss LSM980 Airyscan2 laser scanning confocal microscope (Carl Zeiss Microscopy, White Plains, NY). Single PBMCs were manually selected for recording based on low-resolution preview scans showing only nuclei. All singlet cells were selected in a small neighborhood to avoid biases. In each microscopy session, 24-40 cells were selected for recording in one well for each condition. This was performed in an interleaved manner, capturing 6-8 cells at a time, and then moving to the next well and then repeating this multiple times using the Experiment Designer module for automation. Super-resolution volumes of (358 × 358 × 70 pixels, 0.035 × 0.035 × 0.13 µm/voxel resolution) were recorded in the above-determined positions using Definite Focus autofocusing. A Plan-Apochromat 63 × 1.40 Oil lens, Airyscan2 SR (super-resolution) mode with optimal sampling and frame switching between 3 fluorescence channels to minimize spectral cross-bleed were used. MitoTracker Red, iFluor488, and Hoechs33342 were excited with 561, 488, and 405 nm solid-state lasers, respectively, using the optimal emission filter for each channel. 3D Airyscan2 processing was performed with standard filtering settings. File naming: Four zip files were deposited named as <Donor#id>.zip, where id goes from 1 to 4. Each zip file contains the following Zeiss Microscopy format image files: <Condition>_<Donor#id>_<Stain#batch>_<Cell>.czi <Condition>: 1G – Control culturing in 6-well plates for 25h uG – simulated microgravity culturing for 25h in NASA Rotating Wall Vessels 1G+TLR – as above, with TLR 7/8 agonist (1 μM R848) uG+TLR– as above, with TLR 7/8 agonist (1 μM R848) 1G+CyD– as above, with cytochalasin D uG+CyD– as above, with cytochalasin D 1G+Q – as above, with quercetin 50µM uG+Q – as above, with quercetin 50µM <Donor#id>: 1-4 indicates biological replicates <Stain#batch>: 1-2 indicates experimental replicates of phalloidin staining and imaging session <Cell>: arbitrary number to distinguish images within the same condition/donor/stain set. See image analysis pipelines used with these data at: https://github.com/gerencserlab/Superresolution-actin-and-mitochondria-analysis