Gelatin from porcine skin

Antigen-specific immunoglobulins were quantified by sandwich ELISA. Briefly, high protein binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria) were coated overnight at 4°C with Soluble Total Leishmania infantum Antigens (Silvestre et al., 2008 (link)) prepared in NaHCO3 0.1 M to a final concentration of 40 μg/ml. Plates were then washed with PBS Tween 0.1%, blocked with 1% gelatin from porcine skin (Sigma-Aldrich, MO, USA) in PBS (blocking buffer) for 1 h at 37°C and re-washed. Each serum was then diluted 1:100 in blocking buffer and added to the plates in duplicate. Wells filled with just blocking buffer were used as blanks. Plates were incubated for 2 h at 37°C and re-washed. Afterward, IgG and IgG1 were detected using horseradish peroxidase (HRP) coupled α-mouse antibodies [diluted 1:5,000 (IgG1; Southern Biotech, AL, USA) or 1:8,000 (IgG; Southern Biotech, AL, USA) in blocking buffer; incubated for 1 h, at 37°C]. Plates were washed for the last time, and the substrate (ortho phenyl diamine (OPD) in citrate buffer) was added for 10 min, time after which the reaction was stopped with HCl 3 N. Absorbance values were determined at 492 nm in a Synergy^TM 2 Multi-Mode Reader (BioTek instruments, VT, USA).

Human osteosarcoma U2OS cells, retinal pigment epithelium RPE-1 cells and VC-8 hamster cells, V-C8 cells complemented with human BACs (V-C8 + BRCA2 and V-C8 + BRCA S3291A)^{4 (link)} were cultured in DMEM (41966-029, Life Technologies) supplemented with 10% (v/v) FBS, 100 U ml⁻¹ penicillin, and 100 µg ml⁻¹ streptomycin at 37 °C and 6% CO₂. Patient fibroblasts RAD51-T131P and BJ foreskin fibroblasts (ATCC) were grown in DMEM (41965-039, Life Technologies) supplemented with 15% (v/v) FBS, 100 U ml⁻¹ penicillin, and 100 µg ml⁻¹ streptomycin at 37 °C and 6% CO₂^{8 (link)}. PL2F2 mouse ESCs were maintained in DMEM with 15% fetal bovine serum, 0.00072% β-mercaptoethanol, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, and 0.292 mg ml⁻¹l-glutamine at 37 °C and 5% CO₂^{9 (link)}. ESCs were cultured on feeder cells (mouse embryonic fibroblasts, inactivated with 10 mg ml⁻¹ mitomycin C) for two passages, after they were transferred to feeder-free, gelatinized tissue culture dishes (0.1% gelatin from porcine skin, Sigma).

Sodium percarbonate (SPO) (20–30% H₂O₂), calcium peroxide (CPO) (75% purity), fibrinogen from bovine plasma (type I-S, 65–85% protein; ≥75% of protein was clottable), hyaluronic acid sodium salt (HA) from Streptococcus equi, glycerol, gelatin from porcine skin (gel strength: ~175 g Bloom, type A), thrombin from bovine plasma, aprotinin from bovine lung, and catalase from bovine liver (2000–5000 U/mg) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Gelatin from porcine skin, poly(ε-caprolactone) (PCL with M̄_n = 80 000 g mol⁻¹), a dialysis bag with a cut-off of 12 kDa, Hoechst dye and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT powder) were purchased from Sigma-Aldrich. Poly(ethylene glycol) (PEG, M̄_n = 1000 g mol⁻¹), acryloyl chloride, methacrylic anhydride, sodium hydroxide, diethyl ether, ethanol, and DMSO were obtained from Merck. Ammonium persulfate (APS), potassium dihydrogen phosphate (KH₂PO₄), and ethylenediaminetetraacetic acid (EDTA) were prepared from Fluka. Toluene and acetone were provided by Dr Mojallali Co. Coconut oil as the internal phase was obtained from the market. Distilled water was freshly used for all the experiments.

Polycaprolactone diol (PCL diol, M_w = 2000 Da), and gelatin from porcine skin were purchased from Sigma-Aldrich, Shanghai, China. Dimethyl sulfoxide-d6 (DMSO-d6, 99.8%), stannous octoate (Sn(Oct)₂, purity = 95%), TA (purity = 99%) and 2,2-Bis (hydroxymethyl) propionic acid (DMPA) were purchased from Shanghai Titan Technology Co., Ltd. DMSO (purity = 99.7%) was purchased from Beijing Innochem Science & Technology Co., Ltd, Beijing, China. Hexamethylene diisocyanate (HDI, purity = 98.5%), BDO (purity = 99%) and methylbenzene (purity = 99.5%) were purchased from Guoyao Reagents Company (Shanghai, China). ADP (purity = 98%) was purchased from Beijing Hwrkchemical Co., Ltd., Beijing, China. PT and APTT test kits were purchased from Shanghai Sun Biotechnology Co., Ltd., Shanghai, China. Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Shanghai MesGen Biotechnology Co., Ltd, Shanghai, China. Fetal bovine serum (Gibco) was purchased from Beyotime, Shanghai, China.

To prepare casein gelatin agar plates 1% (w/v) casein sodium salt from bovine milk (Sigma–Aldrich, United States) and 1% (w/v) gelatin from porcine skin (Sigma–Aldrich, United States) were thoroughly dissolved in 0.02 M NaOH and the pH was equilibrated to 7 ± 0.2 as described by Montville (1983) (link). Wherever, the proteolytic inhibition by ethylenediaminetetraacetic acid (EDTA) was tested, EDTA was added to the casein gelatin medium in 1 mM final concentration. Finally, 1.5% (w/v) of agar was added to the casein gelatin medium. The casein gelatin medium was autoclaved at 110°C. 40 ml of the medium was poured into Petri dishes with 9 cm diameter. Wells (6 mm diameter) were cut into the casein gelatin agar using an agar punch cutter.