Four to five week-old C57BL6/J females were injected with 5 IU pregnant mare serum gonadotropin (National Hormone Peptide Program, Torrance, CA) followed 48 hours later by an injection of 5 IU human chorionic gonadotropin (hCG) (National Hormone Peptide Program, Torrance, CA). Following hCG injection, females were placed overnight with a C57BL/6J male of proven fertility, and checked the following morning for the presence of a vaginal plug. Mated females were euthanized, ovaries and oviducts placed in 1 ml of prewarmed M2 medium (Millipore MR-015P) in a 60 mm tissue culture dish, and cumulus-covered one-cell embryos were released by carefully tearing the ampulla open with fine forceps. Hyaluronidase solution (0.3mg/ml, Sigma H3506) was added to medium to free adherent cumulus cells. One-cell embryos were collected via glass transfer pipette, washed in three separate drops of medium, moved to a drop of medium covered in oil, and incubated at 37°C with 5% CO2 for 10–30 minutes before transfer. Pseudopregnant (0.5 days) CD-1 or C57BL/6J females were obtained from matings with vasectomized CD-1 males. Approximately 10 one-cell embryos were transferred via glass pipette to the infundibulum of each oviduct of anesthetized pseudopregnant females.
Hyaluronidase solution
Manufactured by Merck Group
Sourced in Germany
Hyaluronidase solution is a laboratory reagent used to degrade hyaluronic acid, a naturally occurring polysaccharide in the extracellular matrix of various tissues. It acts as a spreading factor, facilitating the diffusion and absorption of injected substances.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Tyrode s salt solution, by Merck Group (2 mentions)
Collagenase a, by Roche (2 mentions)
Btx electroporation system, by Harvard Apparatus (2 mentions)
Btx porator, by Harvard Apparatus (2 mentions)
Mineral oil, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Serva Electrophoresis (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Hyaluronic acid solution, by Merck Group (2 mentions)
Crispr rna crrna, by Merck Group (2 mentions)
Tracrrna05n 5nmol, by Merck Group (2 mentions)
Nepa21 super electroporator, by Nepa Gene (2 mentions)
Lsrii system, by BD (2 mentions)
Collagenase 4, by Merck Group (2 mentions)
Sodium chloride (nacl), by Roche (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Clinical refractometer, by Atago (1 mentions)
Oxoid signal blood culture system, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Tcm 199, by Thermo Fisher Scientific (1 mentions)
19 protocols using hyaluronidase solution
1
Superovulation and Embryo Transfer in Mice
2
In vivo Electroporation for Muscle Fiber Transfection
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Hyaluronidase solution (2 mg/ml) (Sigma-Aldrich) was injected in the hind limb footpad of anesthetized animals. 30 min later, 20 μg of plasmid DNA in 20 μl of 0.9% NaCl solution was injected. One gold-plated acupuncture needle was placed under the skin at heel, and a second one at the base of the toes, oriented parallel to each other and perpendicular to the longitudinal axis of the foot and connected to a BTX Electroporation System (Harvard apparatus). 20 pulses, 20 ms each, 1 s of interval were applied to yield an electric field of 100 V. Single fibers cultures were carried out 7–10 days later.
FDB fibers were isolated 7-10 days after in vivo transfection. Muscles were digested in collagenase A (4 mg/ml) (Roche) dissolved in Tyrode’s salt solution (pH 7.4) (Sigma-Aldrich) containing 10% FBS. Single fibers were isolated, plated on laminin-coated glass coverslips and cultured as reported in the “primary cell cultures” paragraph.
FDB fibers were isolated 7-10 days after in vivo transfection. Muscles were digested in collagenase A (4 mg/ml) (Roche) dissolved in Tyrode’s salt solution (pH 7.4) (Sigma-Aldrich) containing 10% FBS. Single fibers were isolated, plated on laminin-coated glass coverslips and cultured as reported in the “primary cell cultures” paragraph.
3
Quantitative Hyaluronidase Assay Protocol
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The enzymatic assay of hyaluronidase antigenic fragments was determined as U/mg using a quantitative turbid metric method based on Dorfman, A (30 (link)). In principle, hyaluronic acid as substrate is hydrolyzed and depolymerized by hyaluronidase. Conditions of this method are T = 37°C, pH = 5.9, A 600 nm, light path = 1 cm. Briefly, the recombinant protein was dissolved in 1 mL enzyme buffer (0.02 M phosphate buffer, 0.45% NaCl (Roche, Germany), 0.01% bovine serum albumin (BSA) (Sigma, Germany), pH = 6.8-7.0, mixed with 0.5 mg HA (Sigma, Germany), which was dissolved in 1 mL substrate butter (0.3 M KH2PO4/Na2HPO4 (Merck, Germany), pH = 5.30-5.35). Enzyme digestion was allowed to proceed for 45 minutes at 37°C. Turbidity was generated at the end of the incubation by adding 10 mL acid albumin (Merck, Germany) solution. Optical density at 600 nm was determined exactly five minutes after the addition of acid albumin.
National formulary standard hyaluronidase solution (NF Std) (Sigma, Germany) was used as a standard sample in different concentrations and to establish a standard curve; units of activity were calculated by comparing them to this curve. A standard curve was made using commercial hyaluronidase (Sigma, Germany) with different known concentrations diluted from a stock of 400-1000 mg/mL as follows: 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6.
National formulary standard hyaluronidase solution (NF Std) (Sigma, Germany) was used as a standard sample in different concentrations and to establish a standard curve; units of activity were calculated by comparing them to this curve. A standard curve was made using commercial hyaluronidase (Sigma, Germany) with different known concentrations diluted from a stock of 400-1000 mg/mL as follows: 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6.
4
Intramuscular DNA Electroporation in Mice
5
Mouse Zygote Isolation for Microinjection
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Ethical approval for the procedures on animals was obtained from I Local Ethical Commission for Experiments on Animals in Warsaw (decisions no. 527/2013 and 176/2016). Embryos used in all experiments were isolated from F1(C57BL/6×CBA) mouse females, which were induced to superovulate by injection of 10 IU of PMSG (Pregnant Mare Serum Gonadotropin; Folligon, Intervet, Netherlands) and 10 IU of hCG (Human Chorionic Gonadotropin; Chorulon, Intervet, Netherlands) 48–52 h later. Females were mated with males of the same strain immediately after hCG injection. Zygotes were collected from mated females 21–22 h post hCG injection. They were released from the oviducts into the hyaluronidase solution (300 μg/ml, Sigma) to disperse follicular cells. Follicular cells-free zygotes were washed and incubated in drops of M2 medium, under mineral oil (Sigma), at 37.5 °C, 5% CO2 in the air until being used for microinjections60 (link).
6
Inhibition of Hyaluronidase Activity Assay
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The inhibition study of hyaluronidase was set by a turbidimetric method described previously [13 (link),33 (link)]. The assay started by adding 20 µL of tested compound in monosodium phosphate buffer and then 40 µL of hyaluronidase solution (22.5 U/mL, Sigma Aldrich, Poznan, Poland) to the wells of 96-wells plates. The mixture was kept in the dark for 10 min at the temperature 37 °C. After incubation, 40 µL of hyaluronic acid solution (0.03%, Sigma Aldrich) in monosodium phosphate buffer was added to the wells. The mixture was kept in the dark for 45 min at the temperature of 37 °C. Finally, 300 µL of bovine serum albumin (0.1%, Serva) in sodium acetate buffer was added to the mixture and incubated for 10 min at room temperature. Changes in turbidity were measured by a microplate reader (BioTek, Winooski, VT, USA) at 600 nm. Heparin (WZF, Polfa, Warsaw, Poland) was a positive control. The assay was carried out in triplicate. The inhibitory activity of the tested compound was calculated from the equation [13 (link),33 (link)]: where AHA—absorbance of solution without the enzyme (positive control), AHYAL—absorbance of solution without the tested compound (negative control), AAN—absorbance of solution with the tested compound.
7
Synovial Fluid Analysis for Infection
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Synovial fluid samples were obtained by routine aseptic technique. The sample was divided into 2 EDTA blood collection vials and 1 collection in a blood culture bottle (Oxoid Signal blood culture system, Oxoid microbiological products, Thermo Fisher, Hampshire, UK) or in a plain tube if the volume available was <10 ml. Cytology was performed within 12 h on 1 EDTA sample and the following parameters determined: NCC, %N, TP, and presence of intracellular bacteria. The NCC was determined using a Neubauer chamber after treating synovial fluid with hyaluronidase solution (Sigma Aldrich, UK). The other cytological parameters were determined by examination of direct smears and cytospin samples, stained with a modified Romanowsky stain, by a board-certified clinical pathologist. TP was quantified on EDTA samples by a clinical refractometer (Atago, Japan). Bacterial culture was performed on plain samples or blood culture samples using MacConkey and blood agar. Blood culture samples were processed according to the manufacturer's guidelines. The second EDTA sample was frozen for 1–2 months at −20°C until SAA quantification.
8
Plasmid DNA Electrotransfer in Mice
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Hyaluronidase solution (2 mg/ml) (Sigma-Aldrich) was injected in the hind limb footpad of anesthetized animals. 30 min later, 20 μg of plasmid DNA in 20 μl of 0.9% NaCl solution was injected. One gold-plated acupuncture needle was placed under the skin at heel, and a second one at the base of the toes, oriented parallel to each other and perpendicular to the longitudinal axis of the foot and connected to a BTX Electroporation System (Harvard apparatus). 20 pulses, 20 ms each, 1 s of interval were applied to yield an electric field of 100 V. FDB fibers were isolated 7 days after in vivo transfection. Muscles were digested in collagenase A (4 mg/ml) (Roche) dissolved in Tyrode’s salt solution (pH 7.4) (Sigma-Aldrich) containing 10% FBS. Single fibers were isolated, plated on laminin-coated glass coverslips and cultured for 24 h before performing experiments.
9
Enzymatic Digestion of Pig Biopsies
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The enzymatic digestion was tested only on domestic pig samples. For that, the whole biopsies were treated at 37 °C for 2 hours with combined collagenase and hyaluronidase solution (both from Sigma-Aldrich, Darmstadt, Germany). After that, samples were centrifuged, the supernatant was removed, and samples were transferred into homogenizing tubes containing the homogenizing buffer (QIAzol or BME + LB) and homogenized immediately.
10
Hyaluronidase Inhibition Assay by Turbidimetry
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The hyaluronidase inhibition assay was set by turbidimetry according to USP XXII-NF XVII, modified by Piwowarski, to the 96-wells plates test [49 ,50 (link)]. Tested compound solutions were freshly prepared before the assay. The protocol started with the addition of 20 µL of the tested compound in monosodium phosphate buffer and 40 µL of hyaluronidase solution (22.5 U/mL, Sigma Aldrich) to each well. The plate was kept in the dark (10 min, 37 °C). In the next step, 40 µL of hyaluronic acid solution (0.03%, Sigma Aldrich) in monosodium phosphate buffer was added to the wells and the plate was incubated again (45 min, 37 °C). Finally, 300 µL of bovine serum albumin (0.1%, Serva) in sodium acetate buffer was added to the wells and the mixture was kept at room temperature for 10 min. The assay was carried out in triplicate. Changes in turbidity were measured by a microplate reader (BioTek, Winooski, VT, USA) at 600 nm. Heparin (WZF, Polfa, Warszawa, Poland) was the positive control [35 (link)]. The inhibitory activity of the tested compound was calculated from the Equation (1):
where AHA is the absorbance of solution without the enzyme (positive control), AHYAL is the absorbance of solution without the tested compound (negative control), and AAN is the absorbance of solution with the tested compound.
where AHA is the absorbance of solution without the enzyme (positive control), AHYAL is the absorbance of solution without the tested compound (negative control), and AAN is the absorbance of solution with the tested compound.
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