DOI: 10.5061/dryad.7d7wm384t
title: Data from: Cytosolic S100A8/A9 promotes Ca2+ supply at LFA-1 adhesion clusters during neutrophil recruitment

description: S100A8/A9 is an endogenous alarmin secreted by myeloid cells during many
 acute and chronic inflammatory disorders. Despite increasing evidence of
 the proinflammatory effects of extracellular S100A8/A9, little is known
 about its intracellular function. Here, we show that cytosolic S100A8/A9
 is indispensable for neutrophil post-arrest modifications during
 outside-in signaling under flow conditions in vitro and neutrophil
 recruitment in vivo, independent of its extracellular functions.
 Mechanistically, genetic deletion of S100A9 in mice caused dysregulated
 Ca2+ signatures in activated neutrophils resulting in reduced Ca2+
 availability at the formed LFA-1/F-actin clusters with defective β2
 integrin outside-in signaling during post-arrest modifications.
 Consequently, we observed impaired cytoskeletal rearrangement, cell
 polarization and spreading, as well as cell protrusion formation in
 S100a9-/- compared to WT neutrophils, making S100a9-/- cells more
 susceptible to detach under flow, thereby preventing efficient neutrophil
 recruitment and extravasation into inflamed tissue.
description: # Data from: Cytosolic S100A8/A9 promotes Ca2+ supply at LFA-1 adhesion
 clusters during neutrophil recruitment This dataset includes 8 different
 excel files, relevant to the manuscript: "Cytosolic S100A8/A9
 promotes Ca2+ supply at LFA-1 adhesion clusters during neutrophil
 recruitment". [https://doi.org/10.5061/dryad.7d7wm384t](https://doi.org/10.5061/dryad.7d7wm384t) 5 excel files are the datasets of the 5 main figures and are named: FIGURE X_data availability. 3 excel files are the datasets of the 3 supplementary figures and are named: FIGURE X-FIGURE SUPPLEMENT X_data availability. In each excel file there are different sheets, which contain the raw data of every single figure panel and are named after them (e.g. FIG 1A, FIG 3C...) ## Abbreviations ICAM-1=Intercellular Adhesion Molecule 1 PSGL1=P-selectin glycoprotein ligand-1 LFA-1=lymphocyte function-associated antigen 1 Ca2+=Calcium ion ER= endoplasmic reticulum SOCE=store-operated calcium entry ## Descriptions The Data deposited are datasets collected throughout different experiments employing different methodology and analyzed in different ways. Data are all included in 8 different xlsx excel files, 5 of which are representative of the 5 main figures of the article and 3 of which are representative of the supplementary figures of the article. Each excel file in divided into sheets which represent each figure panel and they are named after them. In every excel sheet you can find the the title of the panel and the raw data which were collected to produce the relative graphs. ### Files and variables #### File: FIGURE\_1\_data\_availability.xlsx * FIG 1A: The amount of S100A8/A9 (ng/mL) released by murine WT neutrophils, stimulated with PBS control, E-selectin or lysed with Triton-X * FIG 1C: Rolling Flux Fraction (the flux of rolling leukocytes as a percent of total leukocyte flux), analyzed over 1 minute * FIG 1D: Number of adherent cell/mm2, analyzed over 1 minute * FIG 1E: Number of adherent cell/mm2 for each vessel analyzed and plotted against the shear rate of that particular vessel to observe the correlation effect between shear rate and number of adherent cells * FIG 1G: Number of adherent cell/mm2, before and after extracellular mutant S100A8/A9 application, to check whether the extracellular protein could rescue the adhesion phenotype in S100a9 knockout mice in the cremaster muscle trauma model where stimulation is triggered only via surgical preparation * FIG 1I: Number or extravasated neutrophils analyzed by Giemsa staining in the perivascular tissue, based on nuclear morphology #### File: FIGURE\_1-FIGURE\_SUPPLEMENT\_1\_data\_availability.xlsx * FIG S1B: S100A9 mean fluorescence intensity of WT murine neutrophils in the intravascular vs tissue compartment, analyzed by confocal microscopy and single cell analysis * FIG S1D: CD18 median fluoresce intensity on neutrophil's surface, analyzed by flow cytometry * FIG S1E: CD11a median fluoresce intensity on neutrophil's surface, analyzed by flow cytometry * FIG S1F: CD11b median fluoresce intensity on neutrophil's surface, analyzed by flow cytometry * FIG S1G: CXCR2 median fluoresce intensity on neutrophil's surface, analyzed by flow cytometry * FIG S1H: CD62L median fluoresce intensity on neutrophil's surface, analyzed by flow cytometry * FIG S1I: PSGL1 median fluoresce intensity on neutrophil's surface, analyzed by flow cytometry * FIG S1J: CD44 median fluoresce intensity on neutrophil's surface, analyzed by flow cytometry * FIG S1L: Number of adherent cell/mm2, before and after extracellular mutant S100A8/A9 application, to check whether the extracellular protein could rescue the adhesion phenotype in S100a9 knockout mice in the cremaster muscle TNF-alpha model where stimulation is triggered via TNF-alpha intra-scrotum injection * FIG S1M: Number of transmigrated neutrophils calculated via Ly6G median fluorescence intensity and Flow-count fluorospheres in transwell assays (3 micron pore size) toward CXCL1 gradient under static conditions #### File: FIGURE\_2\_data\_availability.xlsx * FIG 2B: Number of rolling cells / field of view, normalized to the white blood cell count in flow chamber assays analyzed over 1 minute * FIG 2C: Number of adherent cells / field of view, normalized to the white blood cell count in flow chamber assays analyzed over 1 minute * FIG 2D: Number of adherent cells / field of view, normalized to the white blood cell count in flow chamber assays analyzed over 1 minute in presence or absence of extracellular S100A8/A9, to check whether the extracellular protein could rescue the adhesion phenotype in S100a9 knockout mice * FIG 2F: Median fluorescence intensity of fluorescently labelled ICAM-1 bound on neutrophils' surface, as a read-out of β2 integrin activation, after stimulation with PBS control or CXCL1 chemokine by flow cytometry analysis  * FIG 2G: Adherent neutrophils (as % of total inserted cells) were analyzed by fluorescence emission with TECAN microplate reader, after stimulation with PBS control or CXCL1 chemokine and cells were washed and stained  #### File: FIGURE\_3\_data\_availability.xlsx * FIG 3B: Area (in  micrometer square) and perimeter (in micrometers) of adherent neutrophils polarizing on E-selectin/ICAM-1/CXCL1 coated dishes calculated over time, analyzed via bright field microscopy and ImageJ * FGI 3C: Circularity [4pi(area/perimeter^2) = value of 1.0 indicates a perfect circle] and solidity (the area of a particle divided by its convex hull = value of 1.0 indicates a solid object) of adherent neutrophils polarizing on E-selectin/ICAM-1/CXCL1 coated dishes calculated over time, analyzed via bright field microscopy and ImageJ * FIG 3D: Neutrophil tracks (X,Y psoitions) of 43 slices with a frame interval of 5 seconds (time lapses) during crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2), used to create the rose plots through the chemotaxis tool plugin (Ibidi) of ImageJ software * FIG 3E: Accumulated distance in micrometers of neutrophils crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2) * FIG 3F: Directionality (Euclidean distance/accumulated distance) of neutrophils crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2) * FIG 3G: Velocity in micrometer/min of neutrophils crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2) * FIG 3H: Pyk2 western blot analysis of neutrophils stimulated for 5min with PBS control+ICAM-1 or CXCL1 chemokine+ICAM-1, and analyzing the ratio between phosphorylated Pyk2/total Pyk2 * FIG 3I: Paxillin western blot analysis of neutrophils stimulated for 5min with PBS control+ICAM-1 or CXCL1 chemokine+ICAM-1, and analyzing the ratio between phosphorylated Paxillin/total Paxillin #### File: FIGURE\_3-FIGURE\_SUPPLEMENT\_1\_data\_availability.xlsx * FIG S3A: Percentage of adherent neutrophils at increasing shear rates every 30 seconds, as compared to the initial number of seeded cells #### File: FIGURE\_4\_data\_availability.xlsx * FIG 4D: Number of LFA-1 nanoclusters defined by size thresholding of LFA-1 signal (0.05 square micrometers) in neutrophils crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2) at 3 different time points of 1 minute each of analysis, by live confocal microscopy imaging and ImageJ analysis. Links to macro scripts are provided at the end of the file * FIG 4G: Mean fluorescence intensity of Ca2+ signals in the pre-defined LFA-1 nanocluster areas, and normalized to those areas, in neutrophils crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2) at 3 different time points of 1 minute each of analysis, by live confocal microscopy imaging and ImageJ analysis. Links to macro scripts are provided at the end of the file * FIG 4J: Mean fluorescence intensity of Ca2+ signals in the negative LFA-1 nanocluster areas (calculated subtracting the LFA-1 nanocluster areas pre-defined to the whole cell area), and normalized to those areas, in neutrophils crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2) at 3 different time points of 1 minute each of analysis, by live confocal microscopy imaging and ImageJ analysis. Links to macro scripts are provided at the end of the file * FIG 4L: Mean fluorescence intensity of S100A9 in the pre-defined LFA-1 nanocluster areas or in the negative LFA-1 nanocluster areas, in neutrophils crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2). Cells where then fixed, permeabilized and intracellularly stained for S100A9 to quantify its levels by confocal microscopy and ImageJ analysis. * FIG 4N: Number of LFA-1 events/frame where at least 10 LFA-1 nanoclusters are present in a radius of 10 square micrometers, to define the aggregation index of these clusters * FIG 4Q: Mean fluorescence intensity of F-actin signal in the whole cell area, in neutrophils crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2) at 3 different time points of 1 minute each of analysis, by live confocal microscopy imaging and ImageJ analysis. Links to macro scripts are provided at the end of the file #### File: FIGURE\_4-FIGURE\_SUPPLEMENT\_1\_data\_availability.xlsx * FIG S4A: Mean fluorescence intensity of overall basal Ca2+ levels, in neutrophils seeded on Poly-L-lysin coated dishes under static conditions * FIG S4B: Calmodulin western blot analysis normalized to GAPDH * FIG S4D: β-Actin western blot analysis * FIG. S4E: Ca2+ frequency distribution of number of events/min, calculated from overall Ca2+ recordings of neutrophil crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2). Links to macro scripts are provided at the end of the file * FIG. S4F: Ca2+ frequency of number of events/min averaged on mice, calculated from overall Ca2+ recordings of neutrophil crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2). Links to macro scripts are provided at the end of the file * FIG. S4G: Ca2+ frequency distribution of events' duration in seconds, calculated from overall Ca2+ recordings of neutrophil crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2). Links to macro scripts are provided at the end of the file * FIG. S4H: Ca2+ frequency distribution of events' duration in seconds averaged on mice, calculated from overall Ca2+ recordings of neutrophil crawling in E-selectin/ICAM-1/CXCL1 coated flow chambers under flow condition (shear rate 2 dynes/cm2). Links to macro scripts are provided at the end of the file #### File: FIGURE\_5\_data\_availability.xlsx * FIG 5A: INDO-1 mean fluorescence intensity values used to plot the kinetic graphs showing Ca2+ entry in neutrophils after CXCL1 stimulation in Ca2+ free medium (no extracellular Ca2+ entry) * FIG 5B: INDO-1 mean fluorescence intensity values of Ca2+ entry (ER store release only), from baseline to peak levels (peak/baseline) in Ca2+ free medium * FIG 5C: INDO-1 mean fluorescence intensity values of Ca2+ baseline levels in Ca2+ free medium * FIG 5D: INDO-1 mean fluorescence intensity values used to plot the kinetic graphs showing Ca2+ entry in neutrophils after CXCL1 stimulation in Ca2+ supplemented medium (extracellular Ca2+ entry) * FIG 5E: INDO-1 mean fluorescence intensity values of Ca2+ baseline levels in Ca2+ supplemented medium * FIG 5F: INDO-1 mean fluorescence intensity values of Ca2+ entry and extracellular Ca2+ entry (ER store release + SOCE), from baseline to peak levels (peak/baseline) in Ca2+ supplemented medium * FIG 5G: INDO-1 mean fluorescence intensity values from the peak to the peak halflife (peak/ 1/2 peak) as in area under the curve, in Ca2+ supplemented medium ## Code/software Microsoft Excel [https://github.com/Napo93/AG-Sperandio-MACROS](https://github.com/Napo93/AG-Sperandio-MACROS) [https://github.com/marrlab/Spatial_CA#spatial_ca](https://github.com/marrlab/Spatial_CA#spatial_ca) [https://github.com/szarma/Physio_Ca](https://github.com/szarma/Physio_Ca)