---------------------------------------------------------------------------------------------- EXTRAVASCULAR PLATELETS DATASET ---------------------------------------------------------------------------------------------- Data and Code that accompanies: Zindel J. et al, Primordial GATA6 macrophages function as extravascular platelets in sterile injury, Science, March 5, 2011, Vol. 371, Issue 6533, eabe0595. DOI: 10.1126/science.abe0595 This data package contains a copy of the main data files exactly as used for the figures in this paper. In addition, we provide the raw tif files used for the cell tracking and the respective R-Scripts used to visualize these tracking results. Data files are generally coma separated values (csv) files except for flow cytometry data which are stored in a multi-sheet excel file. Please, contact us at joel.zindel@dbmr.unibe.ch if you have suggestions, find errors, inconsistencies, or any other bug in the data or scripts. We would be happy to help you in any case, as far as we can. Please, use and expand on these scripts for visualization of tracking results. The examples presented here use output files generated by TrackMate plug in in ImageJ. We found that combining the two open source programs was very powerful. The underlying idea also works for tracks generated in Imaris which in our opinion works better when tracking is done in three dimensions and/or manual drift correction is necessary. Finally, the peritoneal macrophage gene expression shown in this manuscript is from data sets (GSE131724 and GSE133127) that are publicly available on Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) and we recommend accessing them there. ------------------------------------ CONTENTS ------------------------------------------ ---------------------------------------------------------------------------------------------- Cell counts: Fig. 1, Fig. 3, fig. S4 ---------------------------------------------------------------------------------------------- Data files: - Fig1_cell_counts_over_time.csv: Manual count of cells recruited (i.e. not displacing anymore) to injury. n01=count at 1 minute, n02=count at 2 minutes,...n75=count at 75minutes. Groups are open-pouch model, closed-pouch model and neutrophils as explained in the manuscript (Fig. 1). - Fig3_cell_counts.csv: experiment=experiment name beginning with date experiment was started. m=mouse id within experiment. p=position imaged (varies between 1-4 per mouse, averaged per mouse for figures and stats shown in paper). group=treatment or genetic modification (AB: stained with 4ug/mouse anti-F4/80 antibody in 140ul Saline, Saline: 140ul Saline only, C57: Wild-type control, App: App-/-, Heparin_100: 100U Heparin in Saline, Heparin_400: 400U Heparin in Saline, Heparin_1000: 1000U Heparin in Saline, DMSO: DMSO control for Argatroban, Argatroban: Argatroban, CD9: anti CD9 blocking antibody, Isotype: Isotype control, ICAM: Icam1/2-/-, Gata6: Gata6-/-, CD18_CD29: CD18 and CD19 blocking antibody, Poly_I: Poly(I), Poly_C: Poly(C), C3: C3-/-, Thbs1: Thbs1-/-, Crig:Crig-/-,Hirudin: Hirudin in Saline, C57_aMarco: anti-mouse Marco blocking antibody, C57_Pbs: Pbs instead of Saline for Staining, control for blocking antibodies that were diluted in Pbs, Msr1_Pbs: Msr-/-, aCD162: anti-mouse CD162 blocking antibody, CD36: CD36-/-. For more detail regarding these groups please consult the manuscript figure legends). n30=manual cell count of cells recruited (i.e. not displacing anymore) to injury. damage_area_5min=maximal area of injury at 5 minutes in square micrometers. --------------------------------------------------------------------------------------------- Cell tracking: fig. S1, fig. S2 --------------------------------------------------------------------------------------------- Raw images: - FigS1E_raw_image.tif - FigS1I_raw_image.tif - FigS1J_raw_image.tif - FigS1K_raw_image.tif - FigS2D_raw_image.tif Data files: - FigS1E_spots_in_track_statistics.csv: The particles in FigS1E_raw_image.tif were tracked using TrackMate plugin in ImageJ, the resulting spots in tracks statistics were exported as csv file. - FigS1I_spots_in_track_statistics.csv: The particles in FigS1I_raw_image.tif were tracked using TrackMate plugin in ImageJ, the resulting spots in tracks statistics were exported as csv file. - FigS1J_spots_in_track_statistics.csv: The particles in FigS1J_raw_image.tif were tracked using TrackMate plugin in ImageJ, the resulting spots in tracks statistics were exported as csv file. - FigS1J_damage_center.csv: Contains coordinate of damage to calculate position relative to damage. - FigS1K_spots_in_track_statistics.csv: The particles in FigS1K_raw_image.tif were tracked Imaris due to need for drift correction. The resulting spots in tracks statistics were exported as csv file. - FigS1K_damage_position.csv: Contains coordinates of damage to calculate position relative to damage. - FigS2D_spots_in_track_statistics.csv: The particles in FigS1E_raw_image.tif were tracked using TrackMate plug in ImageJ, then the resulting spots in track statistics were exported as csv file. R-Scripts: - FigS1_S2_particle_tracking.R: Reads data from spot_in_track_statistic files and calculates relative displacement of cells over time. Plots different visualizations of tracking results some of which are shown in the respective supplemental figures. --------------------------------------------------------------------------------------------- Aggregate volume quantification: Fig. 2C --------------------------------------------------------------------------------------------- Data files: - Fig2C_data_clot_growth.csv: Volume of macrophage and platelet aggregate was determined using Imaris workstation and exported as csv file. volume=aggregate volume in micrometer squared. slice=consecutive numbering of images over time. id=experimental id. percent=volume as percent of maximal volume. time=slice times frames per second. --------------------------------------------------------------------------------------------- Aggregometry traces: Fig. 2E, fig. S3C,fig. S3F --------------------------------------------------------------------------------------------- Data files: - Fig2E_mac_platelet_traces.csv: Representative aggregation traces as shown in Fig. 2E and fig. S3C respectively. - FigS3F_neutrophil_traces.csv: Representative aggregation traces as shown in fig. S3F. --------------------------------------------------------------------------------------------- Aggregate size: Fig. 3I,J --------------------------------------------------------------------------------------------- Data files: - Fig_3_I_J.csv: area:area in square micrometer of defect in collagen. uncovered:area in square micrometer without mesothelial coverage. percentcovered=uncovered/area. spot=technical replicate (1-3 per mouse). --------------------------------------------------------------------------------------------- Aggregate size: Fig. 4B --------------------------------------------------------------------------------------------- Data files: - Fig4B_aggregate_size.csv: Maximal aggregate area in square micrometer was measured using ImageJ. id=experimental identification number. area=are in square micrometer. group=either simple or super aggregate. model=intravial microscopy model used to acquire images. AIW: abdominal imaging window. --------------------------------------------------------------------------------------------- Flow cytometry: Fig. 5E, fig. S5E, fig. S8, B and E --------------------------------------------------------------------------------------------- Data files: - Flow_cytometry_data.xls: Excel file that contains counts by flow cytometry as described in the methods section. The data for each respective figure is in a separate tab. --------------------------------------------------------------------------------------------- Adhesion scores: Fig. 5G, fig. S8G --------------------------------------------------------------------------------------------- Data files: - Fig5_adhesion_score.csv: ID=unique animal identification, experiment=experiment name, date_of_birth=date of birth, date_of_surgery=date of surgery, sex=sex, age=age, group=pharmacological treatment and genetic alteration (Abbreviations are consistent with Fig. 5G, please refer to figure legend for further details, pretreatment=pharmacological treatment, genotype=genotype,hygiene_status=hygiene status (SPF: specific-pathogen-free), adhesion score=adhesion score as described in the methods section, lost_to_follow_up=animal had to be euthanized according to regulations.