Disodium edta

Cell–scaffold constructs were lyophilized (−55°C, 0.2 mbar, 4 h) and then incubated in a papain digest (2 mg papain [MP Biomedicals]/g lyophilized sample, 20 mM L-cysteine [MP Biomedicals], 9 mM disodium EDTA [Sigma-Aldrich], and 3.6 M sodium acetate [Sigma-Aldrich]) at 60°C for 10 h. Following centrifugation of the solution (4000 g, 10 min), the supernatant was collected and stored at −80°C. Samples were thawed for 1 min in a 37°C water bath immediately before compositional analysis. All assays were performed according to manufacturer's instructions or published methods for microtiter plate analysis and fluorescence or absorbance quantified with a multiwell plate reader (Synergy HT; BioTek Instruments, Winooski, VT). Composition fold change relative to day 0 was calculated as C_f/C_i (C_i: content immediately after cell loading; C_f: content after 7, 14, or 28 days of culture).

A 10⁷ CFU mL⁻¹C. sakazakii ATCC 25944 suspension was prepared and placed on a microscope slide as described above. The fluorescent in situ hybridization was performed in three main steps: (i) sample fixation/permeabilization, (ii) hybridization and (iii) washing of the unbound probe. The dried 20 μL samples were covered with 30 μL of 4% (wt/vol) paraformaldehyde (Sigma), followed by 50% (vol/vol) ethanol (Fisher Scientific) for 10 min each, and subsequently air dried. The samples were then covered with 30 μL of hybridization solution containing 10% (wt/vol) dextran sulphate (Sigma), 10 mM NaCl (Sigma), 30% (vol/vol) formamide (Sigma), 0.1% (wt/vol) sodium pyrophosphate (Sigma), 0.2% (wt/vol) polyvinylpyrrolidone (Sigma), 0.2% (wt/vol) ficoll (Sigma), 5 mM disodium EDTA (Sigma), 0.1% (vol/vol) Triton X-100 (Sigma), 50 mM Tris–HCl (pH 7.5; Sigma), and 200 nM of EUB338 probe (Panagene). Samples were covered with coverslips, placed in moist chambers, and incubated for 60 min at 61 °C. Subsequently, the coverslips were removed, and the slides were submerged in a prewarmed (61 °C) washing solution containing 15 mM NaCl (Sigma), 1% (vol/vol) Triton X-100 (Sigma), and 5 mM Tris base (pH 10; Sigma). Washing was performed at 61 °C for 30 min, and the slides were subsequently air dried. The samples could be stored (dark) for a minimum 24 h before observation, if needed.

HLA classes I and II typing were performed as described previously.^{21 (link),40 (link)} Anti–HLA classes I and II IgG Abs were tested with a bead–based detection assay. We used the LABScreen Mixed kit (One Lambda; Thermo Fisher Scientific, Waltham, MA), which simultaneously detects classes I and II Abs, and the single antigen bead assays (Single Antigen kit, One Lambda; Thermo Fisher Scientific) to identify HLA classes I and II specificities.^{40 (link)} Before testing, all sera were pretreated with disodium EDTA (final concentration 10 mM, pH 7.4; Sigma–Aldrich, Milan, Italy) to rule out underestimation of Ab mean fluorescence intensity (MFI) strength due to the prozone phenomenon.^{41 (link)} Screening assay results above a cutoff value of 3.0 for the ratio of sample to negative control were considered positive. Single–antigen results above an MFI cutoff value of 1000 were considered positive.
Heat–inactivated patient sera were tested with C1qScreen (One Lambda; Thermo Fisher Scientific) for identification of complement–binding Abs, as described.^{27 (link)} Ab positivity was assigned at >500 MFI. Serum samples were analyzed in a blinded fashion for the presence of C3d–binding DSAs with the single–antigen flow bead technology, according to the manufacturer’s protocol (Immucor Lifecode Transplant Diagnostics, Nijlen, Belgium).^{26 (link)}