## 4 Materials and Methods
### Binding of Peptides to Cellular GAGs  
The binding of four different TAMRA-labeled peptides (KKAK-2 mer, KKQK-3 mer, KKKQQK-2 mer and CXCL9(74-103)) to cellular GAGs on pgsB-618 clone 16 (HS\({}^{-}\)CHO cells) and pgsB-618 clone 19 cells (HS\({}^{+}\)CHO cells) was assessed using flow cytometry. Since each peptide contains exactly one TAMRA-group connected to the N-terminus, this allows us to quantify the peptides by their fluorescence or absorbance. First, the medium was discarded, the cells were washed with PBS and were detached with trypsin/EDTA at 37 \({}^{\circ}\)C. Subsequently, the cells were resuspended in medium and left at room temperature for 45 min, and afterwards on ice for 15 min. After centrifugation at 177 g and 4 \({}^{\circ}\)C for 7 min, the pellet was resuspended in PBS and the cells were kept on ice. For each condition, 1 \(\times\) 10\({}^{5}\) cells in 100 \(\mu\)L PBS were used and 4.7% (\(v/v\)) FcR blocking reagent and 0.06% (\(v/v\)) Zombie Aqua fixable viability dye were added to the cells. Following incubation in the dark at room temperature for 20 min, a wash step with flow cytometry buffer was performed. Afterwards, 0.24 nmol TAMRA-labeled peptide was added to the cells. After incubation in the dark on ice for 30 min, a wash step with flow cytometry buffer was performed and, subsequently, the cells were fixed with 0.4% (\(v/v\)) formaldehyde in flow cytometry buffer and kept at 4 \({}^{\circ}\)C. Flow cytometric analysis was performed within the following 24 h using a BD LSRFortessa X-20 flow cytometer (BD Biosciences; Franklin Lakes, NJ, USA), and the results were analyzed using FlowJo software (BD Biosciences). Similarly, the binding of three TAMRA-labeled peptides (KKQK-3 mer, KKKQQK-2 mer and CXCL9(74-103)) to cellular GAGs on pgsB-618 clone 16 (HS\({}^{-}\)CHO cells) and pgsB-618 clone 19 cells (HS\({}^{+}\)CHO cells) in the presence of added GAGs was assessed using flow cytometry. In this experiment, different doses (0.03, 0.1, 0.3, 1 or 3 \(\upmu\)g) of heparin (heparinlow in calcium; Iduron; Cheshire, UK), HS (GAG HS01 BN1; Iduron; Cheshire, UK) or HA (70-120 kDa; HA sodium salt 96144; Sigma-Aldrich; St. Louis, MO, USA) were added to the cells, simultaneously with 0.24 nmol of TAMRA-labeled peptide (vide supra). Flow cytometry data (Figures 7C and S2) are represented as median. Statistical analysis of these data consisted of a Shapiro-Wilk test to assess the normality of the different groups. As some groups did not pass the normality test, a nonparametric Kruskal-Wallis test was subsequently performed to evaluate if the median values of the groups varied significantly. In the next step, either a Mann-Whitney test (for unpaired data) or a Wilcoxon test (for paired data) was used to compare the groups. Statistical analysis was performed using GraphPad Prism 8.3.0 software (GraphPad Software; San Diego, CA, USA). The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/ijms23010447/s1](https://www.mdpi.com/article/10.3390/ijms23010447/s1)]([https://www.mdpi.com/article/10.3390/ijms23010447/s1](https://www.mdpi.com/article/10.3390/ijms23010447/s1)). Software, A.K. ; formal analysis, H.C., L.A., E.G., S.C., E.V., N.P., L.V., T.G. and P.P. ; investigation, H.C., L.A., E.G., S.C., E.V., T.G., A.K. and P.P. ; data curation, H.C., T.G., A.K. and P.P. ; writing--original draft preparation, H.C.; writing--review and editing, H.C., L.A., E.G., S.C., E.V., N.P., L.V., S.S., T.G., A.K. and P.P. ; supervision, A.K. and P.P. ; funding acquisition, A.K. and P.P. All authors have read and agreed to the published version of the manuscript. This research was funded by KU Leuven, C1 grant number C16/17/010. [PERSON] obtained an SB PhD fellowship at FWO-Vlaanderen (grant number 1S50018N) and [PERSON] obtained a fundamental PhD fellowship at FWO-Vlaanderen (grant number 11A4220N). Not applicable. All data are included in the text and Supplementary Materials of the manuscript. We thank [PERSON] for providing the pgsB-618 CHO cells.