Hyaluronidase from bovine testes

We collected both oocytes and CCs from 8- to 10-week-old B6D2F1 female mice by superovulation. Superovulation was induced by sequentially injecting 7 IU of PMSG and 5 IU of hCG (San-Sheng Pharmaceutical, China) at an interval of 48 h. Then, cumulus–oocyte complexes were collected from oviducts 14 h after hCG injection and treated with hyaluronidase from bovine testes (Sigma, St. Louis, MO, United States) to obtain dissociated CCs and oocytes.
The oocytes were enucleated in a chamber containing oil-covered HCZB supplemented with 5 μg/ml of CB (Sigma) by Piezo-driven pipette (PrimeT 130 each) of an Olympus inverted microscope (Tokyo, Japan). The nuclei of donor CCs were transferred into enucleated oocytes by direct injection and activated through 5 h incubation in Ca²⁺-free CZB containing 1 mM SrCl₂ and 5 μg/ml CB. The reconstructed embryos were thoroughly washed and cultured in G1 medium under 5% CO₂ at 37°C.
For NT with the HDACi treatment, Scriptaid (Sigma, United States) was employed for a total of 10 h with a concentration of 5 nM by adding to the culture medium at the beginning of zygote activation.

Female mice were superovulated by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG; Sigma-Aldrich, G-4877) followed by 5 IU human chorionic gonadotropin (hCG; Life Sciences, 367222–1000IU) 48 h later. They were subsequently mated with male mice of the same strain and examined the following morning for vaginal plugs to confirm that mating had occurred. Female mice were euthanized by cervical dislocation approximately after 16–18 h of post hCG injection. Oviducts were dissected and pooled into HEPES-buffered F-10 media (Gibco, 11550-043) with 10% serum synthetic substitute (SSS; Irvine Scientific, CA 92705. USA). Zygotes were isolated by tearing the ampulla of oviducts, and cumulus cells were removed using hyaluronidase from bovine testes (Sigma-Aldrich). Collected embryos were cultured in respective media.

Cells were isolated from the skin as described previously (5 (link)). Briefly, shaved dorsal skin was harvested, minced, and digested for 45 minutes at 37°C with 2.0 mg/ml collagenase XI from Clostridium histolyticum (Sigma-Aldrich), 0.5 mg/ml hyaluronidase from bovine testes (Sigma-Aldrich), and 0.1 mg/ml DNAse (Sigma-Aldrich). Single cells were washed with 10% cRPMI, filtered through a 100 μm filter, and stained for flow cytometry staining as described above. For samples to be used for ICS, Brefeldin A was added to the digestion buffer and surface stain cocktail. For enrichment of antigen-specific T cells from skin, we used PE-conjugated KJ1-26 antibody and a PE positive selection kit (Miltenyi biotech).

Nitrotetrazolium blue chloride (NBT), xanthine, xanthine oxidase, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), potassium persulfate, 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (ferrozine), iron (II) chloride tetrahydrate (FeCl₂ × 4H₂O, 1,3,5-Tri(2-pyridyl)-2,4,6-triazine (TPTZ), iron (III) chloride (FeCl₃), aluminum chloride (AlCl₃), potassium acetate, Folin−Ciocalteu reagent, sodium nitrite, sodium molybdate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, 2(3)-t-Butylhydroquinone monomethyl ether (BHA), 2(3)-t-Butyl-4-hydroxyanisole, hyaluronic acid (IV), escin, hyaluronidase from bovine testes, hexadecyltrimethylammonium bromide (CTAB), L-tyrosine, koji acid, and tyrosine from mushrooms were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The standards of eleutheroside B ≥98.0% (HPLC), eleutheroside E ≥98.0% (HPLC), eleutheroside E1 ≥98.0% (HPLC), protocatechuic acid ≥97%, p-hydroxybenzoic acid 99%, vanillic acid ≥97%, caffeic acid ≥98%, and ferulic acid ≥99% were also purchased from Sigma-Aldrich. Solvents used for extraction were purchased from Avantor Performance Materials (Gliwice, Poland).

The collagenase was supplied by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The elastase was obtained from Beijing Coolaber Technology Co., Ltd. (Beijing, China). The hyaluronidase from bovine testes and the fluorogenic MMP 2 substate (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) were bought from Sigma-Aldrich (St Louis, MO, United States). The 10 kDa cutoff centrifugal ultrafiltration filters (YM-10) were purchased from Millipore Co., Ltd. (Bedford, MA, United States). The membranes (0.22 μm) were bought from Tianjin Jinteng Experiment Equipment Co., Ltd. (Tianjin, China). Epigallocatechin gallate was bought from Shanghai Meryer Chemical Technology Co., Ltd. (Shanghai, China). Acetonitrile (ACN) and formic acid (FA) of HPLC grade were purchased from TEDIA Company Inc. (Fairfield, Ohio, USA). The ultrapure water for LC-MS analysis and pure water for the sample process were prepared using the EPED water system (Nanjing EPED Technology Development Co., Ltd, Nanjing, China). All other chemicals were of analytical grade and supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), including methanol (MeOH), hydrochloric acid (HCl), sodium dihydrogen phosphate (NaH₂PO₄), dibasic sodium phosphate (Na₂HPO₄), and ascorbic acid.