Phosphate buffered saline (pbs)

Ten µg of TGM-1 full length (D1/2/3/4/5), D1/2/3, and D4/5 was incubated with EZ-Link™ Sulfo-NHS-LC-Biotin (Pierce, 21335) for 30 min at RT°, and the reaction was stopped by the addition of 50 mM Tris-HCl pH 7.4. The reaction was purified using a G-25 column (GE health, 28922529). For the pulldown, cells were washed with PBS and then incubated with biotinylated protein(s) for 3 h on ice. After incubation, the plates were washed 3× with PBS (Fresenius Kabi) and the cells were harvested in 1× Cell Lysis Buffer (Cell Signaling Technologies, 9803). After spinning, the supernatant was incubated with Neutravidin beads (Pierce, 29201) for 1 h at 4 °C (rotating). Beads were washed 4× using lysis buffer and 3× using 50 mM ammonium bicarbonate (Sigma-Aldrich, 09830), with fresh LoBind tubes used for each wash (Eppendorf, 0030 108.116). The beads were resuspended in 250 µL of 50 mM ammonium bicarbonate with 250 ng of mass spec grade trypsin (Promega V5113), incubated overnight at 37 °C (with agitation), after which peptides were recovered from the beads with a prewashed 0.4-µm filter (Ultrafree MC HV, Millipore, UFC30HV00) and subjected to mass spectrometry analysis.

Borrelia burgdorferi senso stricto strain B31 and wildtype, OspC-deficient and OspC-complemented B. burgdorferi strain 297 (courtesy of Erol Fikrig, Yale University, New Haven, CT, USA) [12 (link)] were cultured in modified Kelly-Pettenkofer medium (AMC, Amsterdam, Netherlands) plus sodium bicarbonate supplemented with 6% rabbit serum (Sigma-Aldrich, Zwijndrecht, Netherlands) [13 (link)] at 33°C until cultures reached mid-late log growth phrase. Spirochetes were assessed for motility, quantified by dark-field microscopy and recovered by centrifugation at 4000xg for 8 minutes and resuspended in PBS (Fresenius Kabi, Graz, Austria) (ex vivo experiments) or RPMI (in vitro experiments). Spirochetes were passaged no more than 5 times. To test spirochetes viability 72 hours post-inoculation, 10 μl biopsy medium was cultured in 7 ml modified Kelly-Pettenkofer medium and inspected by dark-field microscopy.

Poloxamer 407 (BASF, the Netherlands), PBS (phosphate buffered saline, 10 mM phosphate, pH 7.4, 140 mM NaCl, Fresenius Kabi, the Netherlands), IMDM (Iscove's modified Dulbecco's medium, Lonza, Belgium), human elastase (EPC, Missouri, USA), cytochalasin B (Sigma, Germany), N-formyl-Met-Leu-Phe (fMLP; Sigma, Germany). PTG (PBS supplemented with 0.1% Tween 20 and 0.2% gelatin A), TMB (3,3′,5,5′ tetramethylbenzamidine, Uptima INTERCHIM, France), streptavidin horseradish peroxidase (HRP) (Amersham Life Science, UK), 96-well maxisorb plate (Nunc, Denmark), 96-well polysorb plate (Nunc, Denmark). Biacore buffer HBS-PE (GE Healthcare, the Netherlands). Ampex red (Molecular Probes/Thermo Fisher Scientific, US), HRP (Sigma, Germany).

Cells were cultured and restimulated with PMA and ionomycin. B cells were washed twice with cold (MACS) buffer containing PBS (Fresenius Kabi) with 0.5% Albuman (40 g/l) (Sanquin) and 2mM EDTA (Merck). B cells were then resuspended in 80 μl cold B cell medium per 10⁷ total cells and incubated with 20 μl IL-10 Catch Reagent (IL-10 secretion assay, Miltenyi Biotec) per 10⁷ total cells for 5 min on ice. Subsequently, warm (37°C) B cell medium was added at a concentration of 10⁶ cells/ml and cells were kept for 45 min at 37°C under slow continuous rotation. B cells were washed twice with cold (MACS) buffer, resuspended in 80 μl cold (MACS) buffer per 10⁷ total cells and incubated with 20 μl IL-10 Detection Antibody (PE) (IL-10 secretion assay, Miltenyi Biotec) per 10⁷ total cells for 10 min on ice. Next, B cells were washed with cold buffer and resuspended in PBS containing 1% BSA (Sigma-Aldrich). IL-10⁺ and IL-10⁻ B cells were analyzed on a LSRII flow cytometer (BD Biosciences) or isolated by FACS sorting on a FACS Aria (BD Biosciences).

Mice were euthanized with CO₂ after a two-week period of daily oral gavages with curdlan (10 mg/mL) or vehicle. Peritoneal macrophages were isolated by flushing peritoneum with sterile PBS (Fresenius Kabi, Huis Ter Heide, the Netherlands) and 5 mM UltraPure^TM EDTA (Fisher Emergo B.V, Landsmeer, The Netherlands). Cells were plated at a concentration of 2 million cells/mL on a 24-well tissue culture plate (VWR International BV, Amsterdam, The Netherlands), stimulated with RPMI-1640 medium, which contains L-glutamine and 25 mM HEPES (Thermo Fisher Scientific, Bleiswijk, The Netherlands). We also added the supplements of 10% (v/v) fetal bovine serum (FBS) (Bodinco BV, Alkmaar, The Netherlands) and 1% (v/v) penicillin–streptomycin (Fisher Emergo B.V, Landsmeer, The Netherlands). Cells were incubated for 24 h at 37 °C with 5% CO₂ to let the peritoneal macrophages adhere. Subsequently, the medium was aspirated and fresh medium, or fresh medium with 10 ng/mL lipopolysachcharide (LPS) (Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands), was added to the macrophages for 24 h. Thereafter, macrophages were lysed in Tripure isolation reagent (Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) for RNA extraction and the supernatants were stored to measure cytokines.

To induce cognitive impairment, mice were injected with PBS or cisplatin in PBS (Fresenius Kabi USA, Lake Zurich, IL) intraperitoneally (IP) at a dose of 2.3 mg/kg daily, for five days followed by a five-day rest period and another five days of cisplatin injection. Freshly isolated mitochondria were delivered to the mice nasally at 48 and 96 h after the last dose of cisplatin or PBS. To enhance the permeability of the nasal mucosa, 50 U of hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) in 3 µL was administered per nostril (total of 100 U per mouse) 30 min prior to the nasal administration of mitochondria. For each mouse, up to 170 µg of mitochondria isolated from 4.5E6 human MSC in a total volume of 12 µL were administered at 3 µL per nostril in alternation (total volume 12 µL/mouse).