Phosphate buffered saline (pbs)

To analyse the purity and efficiency of the B and Th cell isolation, flow cytometry analysis was performed. In order to identify B and Th cells, we stained for CD3 (total T cells), CD4 (Th cells) and CD19 (B cells) using fluorophore-conjugated antibodies (Table 1). To control for background signalling and to set the gates, isotype control antibodies were used. The cells were stained in staining buffer [PBS (BioConcept), 2% FBS (Gibco)] and subsequently fixed with 4% paraformaldehyde (Sigma) for 15 min. The cells were analysed with the BD FACSCanto ll flow cytometer (BD Bioscience). Data was processed by the BD FACSDiva software (BD Bioscience). The data was analysed using the FlowJo software (Treestar, Inc.).Table 1

List of fluorophore-conjugated antibodies

Target	Fluorophore	Clone	Dilution	Company
CD4	FITC	OKT4	1:80	eBioscience
CD19	PE	HIB19	1:20	eBioscience
CD3	APC	UCHT1	1:80	Biolegend

The solutions were prepared 1 to 3 days before electrospinning. For each scaffold, a polyethylene glycol (PEG) (35 kDa, Sigma-Aldrich, Berlin, Germany #81310) solution was prepared by adding 1.5 g of PEG and 3.5 g of chloroform (Sigma–Aldrich, Germany, #132950). The DP solution was produced by adding 0.6 g of DP powder, 3.52 g of chloroform, and 0.88 g of 1,1,1,3,3,3-Hexafluoro-2-propanol (HFP, Sigma-Aldrich, Germany, #105228) into a glass with a screw cap. For the incorporation of recombinant human IGF-1 (PeproTech, Boston, MA, USA, #100-11-100UG), a total amount of 4 µg of IGF-1 dissolved in 400 µL of phosphate-buffered solution (PBS, BioConcept, Allschwil, Switzerland, #3-05F39-I) containing RSA (10 µg/mL IGF-1 and 0.25% RSA in PBS) was added drop-wise to the DP solution, while stirring for five minutes on the magnetic stirrer at 500 rpm. The solution was shortly mixed on the vortex and further emulsified in an ultrasonic bath for 15 min. The emulsion was filled in a 5-mL glass syringe (Huberlab, Aesch, Switzerland,# 3.7102.33) and used immediately for electrospinning. As this protocol for water-in-oil emulsion with IGF-1 was the same as used previously for PDGF-BB incorporation, homogenous distribution of water droplets containing IGF-1 within the DP fibers can be assumed [23 (link)].

The EAI assay was performed as described in detail elsewhere [9 (link)]. In brief, erythrocytes obtained from Balb/cJRj mice were purified using a Ficoll gradient (Ficoll-Paque, VWR, Cat#17-1440-02) and were stored for up to 2 weeks in DMEM (Gibco, Cat#61965) containing 10% FCS FCS (Amimed, Cat#2-01F30I) at 4°C prior to use.
Serial dilutions of sera were performed in 96-well U-bottom plates (Greiner, Cat#650161) in DMEM containing 10% FCS. 5x10⁵ cfu B. taylorii expressing GFP were added per well and the plates were incubated at 35°C, 5% CO₂ for 1h prior to the addition of 10⁶ red blood cells (multiplicity of infection, MOI = 0.5) in 100 μl DMEM containing 10% FCS. The next day, the supernatant was removed, the red blood cells were fixed using 1% PFA (EMS, Cat#EMS-15710) and 0.2% gluturaldehyde (EMS, Cat#16020) in PBS (BioConcept, Cat#3-05F29-I) for 10 min at 4°C in the dark. After quenching with 2% FCS in PBS, the cells were analysed for GFP signal by Flow Cytometry (BD Canto II using HTS autosampler).