Pbs solution

Cell pellets were suspended in 0.25 mL 1X PBS solution (Sigma-Aldrich) and counted using an automated cell counter (Scepter, Millipore Corporation, Billerica, MA, USA).
Free hemoglobin concentration was assessed by applying the Allen correction to absorbance readings at 563 nm, 577 nm, 600 nm (Synergy HTX).

PBS solution, acrylamide, N-ethylmaleimide, DTT, GSSG, eosin isothiocyanate, and 19 different flavonoids (quercetin, 3-O-methyl quercetin, isoquercetin, quercitrin, rutin, morin, rhamnetin, isorhamnetin, fisetin, apigenin, apigenin-7-glucoside, luteolin-7-glucoside, kaempferol, eupatorin, eupatorin-5-methyl-ether, genistein, naringenin, cyanidin, and 6,2′,4′-trimethoxyflavone) were purchased from Sigma-Aldrich, EDTA (0.5 M solution pH 8.0) from IBI Scientific. SYPRO Orange was from Invitrogen.

Poly(ethyleneglycol)methyl ether, thionyl chloride, silver nitrate, sodium borohydride, 3-chloro-1-propanethiol, PBS solution, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin, p-nitroso-N,N’-dimethylaniline, hydrochloric acid (≥37%), tetrahydrofuran, chloroform, ethanol, triethylamine, water (LC-MS grade), acetone, acetone-d6 and trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene] malononitrile were purchased from Sigma-Aldrich (Merck Group, Milan, Italy).
The 5,10,15,20-[p-(ω-methoxy-polyethyleneoxy)phenyl]-porphyrin (P(PEG750)₄) was obtained as reported elsewhere [39 (link)].

Flow cytometry was used to find out the percentage of monocytes and DCs that express TLR2. The process used for sampling was as follows: for each participant, a whole blood sample of 100 μL and fluorochrome-conjugated monoclonal antibodies underwent incubation in the dark for 20 min against these antigens:□

CD1c (BDCA-1) FITC/Pacific Blue anti-Human Lineage Cocktail (anti-CD3, CD14, CD16, CD19, CD20, CD56)/TLR2 PE (Biolegend, San Diego, CA, USA);

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BDCA-2 FITC/CD123 Pe-Cy7/CD45 V450/TLR2 PE (Biolegend);

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CD14 FITC/CD16 V450/HLA-DR Pe-Cy7 (BD Biosciences, San Jose, CA, USA) and TLR2 PE (Biolegend).

Thereafter, Lysing Buffer (BD Pharm Lyse) was used to treat the samples, and they were then washed in PBS solution (Sigma-Aldrich, St. Louis, MO, USA). The Cytoflex LX (Beckman Coulter, Brea, CA, USA) was used to collect the samples. The data were analyzed using the Kaluza Analysis software and evaluated with dot plots. The definition of mDCs was BDCA1+ Lin− cells; plasmacytoid DCs, BDCA2+ CD123+ cells; classical monocytes, CD14+ CD16− cells; and non-classical monocytes, CD14+ CD16+. A gating step with HLA-DR was added to improve the monocyte purity and isotype controls (Biolegend) used for the determination of binding that was unspecific.

Graphite powder, TEOS, PBS solution, HCl, H₂SO₄, absolute ethanol, glacial acetic acid, nutrient broth, and nutrient agar were purchased from Sigma Aldrich, Malaysia. These chemicals were analytically graded and used without any purification.
Preosteoblast (MC3T3-E1) cell lines and alpha-MEM (α-MEM) were supplied by ATCC and Hyclone Laboratories Inc., respectively. Fetal bovine serum (FBS) and l-glutamine penicillin/streptomycin were purchased from ThermoFisher Scientific. Male albino mice (BALB/c) weighing 23–25 g (aged 5–7 weeks) were supplied by the National Institute of Health. Approval from the Animal Ethical Committee was obtained to carry out experiments on mice.

DRG neurons were dissociated and cultured as described previously^{25 (link), 26 (link)} with modifications. Briefly, the L4-L6 DRGs were removed from the adult SD rats, and transferred to Ca²⁺/Mg²⁺-free Hibernate A (BrainBits, Springfield, IL), where the axon roots and dural tissue were manually removed. The DRGs were then transferred to 0.1% collagenase type I (Sigma, St Louis, MO). Following 1.5 h incubation at 37 °C, the DRGs were dissociated in 0.25% trypsin (Gibco) for an additional 15 min at 37 °C, and mechanically triturated through a pipette into the single cell suspension. To remove SCs, a partial purification step was performed by centrifugation at 900 rpm for 5 min on 15% BSA in PBS solution (Sigma). The obtained DRG neurons were cultured on the coated plates in Neurobasal-A and B-27 minus insulin (Gibco) supplemented with penicillin–streptomycin (both 50 U/ml, Gibco). The modulatory effects of IGF-1 on neurite outgrowth were treated with IGF-1 (25 ng/ml; R&D Systems) for 48 h. For the pre-lesion injury assay, we transected the sciatic nerve, waited 4 days, and then cultured adult DRG neurons for 72 h to evaluate their regeneration capacity.

The percentage of peripheral lymphocytes expressing TLR2 was measured. Blood samples of 50 µL with the following antibodies were used:□

CD19 FITC/CD3 PE;

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CD4 FITC/CD8 PE/CD3 PerCP;

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CD3 FITC/CD16 PE/CD56PE;

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TLR 2 PE/CD4FITC;

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TLR2PE/CD8FITC;

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TLR2PE/CD19FITC (BD Biosciences, San Jose, CA, USA).

Thereafter, the samples were treated with Lysing Buffer (BD Pharm Lyse) and PBS solution (Sigma Aldrich) for washing. We then collected the samples using a Cytoflex LX (Beckman Coulter) and analyzed the data using the Kaluza Analysis program. The data were evaluated with dot plots. Isotype controls (Biolegend) were used to determine unspecific binding.
We also calculated the percentages of the following: T helper cells (CD3+ CD4+), T cytotoxic cells (CD3+ CD8+), B lymphocytes (CD3− CD19+), natural killer cells (CD3− CD16+ CD56+) and natural killer T-like cells (CD3+ CD16+ CD56+). Using forward and lateral dispersions and a two-color fluorescence plot, we established the population of target cells. Gates were placed around individual cell populations to determine the relative CD45+ cell percentages. To determine the background signal and to exclude contamination and cell aggregates, FITC IgG1 κ and PE IgG1 κ were used as Isotype controls.