Colon samples were obtained from slaughterhouse material of approximately 10-week-old piglets. A 20 cm section of mid-sigmoid colon was obtained immediately after slaughter, and intestines were placed in aerated Krebs Henseleit Buffer containing 5 mM glucose (hereafter referred to as modified-KHB, K3753, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2.5 g/L Bovine serum albumin (BSA, A7906, Sigma-Aldrich, St. Louis, MO, USA). After this, the intestines were flushed with modified-KHB. Then, they were inverted, and a sac was created using dialysis clamps by filling them with modified-KHB. The sacs were incubated for 20 min in Ca2+-free KHB buffer containing 20 mM EDTA and 10 mM DTT in a shaking 37 °C water bath. Following this washing step, intestines were re-verted and filled with an isolation buffer containing Ca2+-free KHB buffer, 2.5 g/L BSA and 400 U/mL hyaluronidase type IV (3884, Sigma-Aldrich, St. Louis, MO, USA). After a fifteen-minute incubation, the intestines were gently massaged and cells were collected, washed and counted using a Cellometer K4 (Nexcelom Bioscience, Lawrence, MA, USA), and viability was simultaneously assessed by staining with ViaStain (CS2-0106, Nexcelom Bioscience, Lawrence, MA, USA). Cells were taken up in KHB medium containing 1 mM HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid) and 2.5 mM glucose, pH 7.4.
Hyaluronidase type 4
Manufactured by Merck Group
Sourced in United States
Hyaluronidase type IV is a laboratory reagent used to break down hyaluronic acid, a component of the extracellular matrix. It facilitates the dispersal and absorption of other injected substances.
Automatically generated - may contain errors
Lab products found in correlation
Model sd9, by Natus (3 mentions)
Model tds 220, by Tektronix (3 mentions)
Collagenase type 2, by Thermo Fisher Scientific (2 mentions)
Matrigel, by BD (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
K3753, by Merck Group (1 mentions)
N nitrosodimethylamine, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Vectashield hard set mounting medium containing dapi, by Vector Laboratories (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
70 μm nylon cell strainer, by BD (1 mentions)
Agarose, by Merck Group (1 mentions)
Veriquest sybr green qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Pcr master mix, by Thermo Fisher Scientific (1 mentions)
Lb agar, by HiMedia (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by HiMedia (1 mentions)
X gal, by HiMedia (1 mentions)
11 protocols using hyaluronidase type 4
1
Isolation of Piglet Colonic Epithelial Cells
2
Muscle Fiber Transfection in Adult Mice
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Transfection of plasmids in FDB muscles of adult mice was performed as described (DiFranco et al., 2009 (link)). In brief, mice were anesthetized using 5% isoflurane, footpads were injected with 2 mg/ml hyaluronidase type IV (Sigma-Aldrich), and 1 h later, ~20 μg total cDNA was injected (10 μl) followed by electroporation using acupuncture needles as electrodes and delivering 20 pulses of 60 V and 30-ms duration at 1 Hz using a square pulse stimulator (model SD9; Grass Technologies) visualized with an oscilloscope (model TDS 220; Tektronix). Six–10 d after electroporation, mice were euthanized and FDB muscles were harvested. Muscles were digested using 4 mg/ml collagenase type 2 (16101015, ThermoFisher) for 1 h at 36°C under agitation, followed by mechanical dissociation, adapted from Casas et al. (2010) (link). Isolated fibers were plated onto Matrigel (BD Biosciences)-coated coverslips in DMEM medium supplemented with Pen/Strep and 10% horse serum (HS), and used within 24 h after plating.
3
Bacterial Transformation of pMar4s Plasmid
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The pMar4s plasmid was transformed into SS2 and SEZ by electroporation27 (link)28 (link). Briefly, the optical density of the bacterial culture medium at 600 nm (OD600) was monitored with a spectrophotometer. SS2 was cultured in THB with the addition of 40 mM DL-threonine to an OD600 = 0.4. SEZ cells were prepared similarly but were treated with hyaluronidase Type IV (Sigma, USA) (45 U/ml for 30 min) to allow efficient pelleting. The cells were harvested by centrifugation at 10,000 × g, the supernatant was discarded, and the pellets were washed twice in 20 mL of 0.5 M sucrose solution. Finally, the cells were resuspended in 250 μL of a 10% glycerol and 0.5 M sucrose solution, and divided into aliquots. The competent cells were stored at −80 °C.
Approximately 1 μg of the pMar4s plasmid was added to the competent cells. The electroporation parameters were as follows: 25 kV/cm, 200 Ω and 25 μF. Immediately after electric shock, pre-warmed THB media containing 0.3 mmol glucose (1 mL) was added to the cuvette and incubated at 28 °C for 3–4 h.
Approximately 1 μg of the pMar4s plasmid was added to the competent cells. The electroporation parameters were as follows: 25 kV/cm, 200 Ω and 25 μF. Immediately after electric shock, pre-warmed THB media containing 0.3 mmol glucose (1 mL) was added to the cuvette and incubated at 28 °C for 3–4 h.
4
Isolation of Rat Adipose-Derived Cells
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N-nitroso-dimethylamine, collagenases type I, hyaluronidase type IV, DNase I, penicillin and streptomycin were purchased from Sigma-Aldrich Co. (St. Louis, MO). Growth media and gentamicin were obtained from GIBCO BRL., Life Technologies (Gaithersburg, MD). Collagenase Medium consisted of RPMI 1640 medium containing 0.1% collagenase type I, 0.01% hyaluronidase type IV, 0.01% DNase I, 100 U/ml penicillin and 100 µg/ml streptomicin. Bovine serum albumin (BSA) was the product of Fermentas International Inc. (Burlington, Canada). Polyclonal rabbit anti-rat-GLUT-1, anti-rat-GLUT-3 and anti-TGF ß-1 were bought from Abcam Inc. (Cambridge, MA, USA). Texas red-conjugated anti-rabbit secondary antibody and Vectashield Hard Set mounting medium containing DAPI were from Vector Laboratories, Ltd. (Peterborough, England).
Phospate buffered saline (PBS) contained: 140 mM NaCl, 5 mM KCl, 8 mM Na2HPO4 at pH 7.3. Phosphate buffered saline with Tween (PBST) consisted of 0.1% Tween 20, 20 mM Na2HPO4, 115 mM NaCl; pH 7.4. The collagenase Solution for perfusion contained 30 mg collagenase type IV in 100 ml PBS solution.
Phospate buffered saline (PBS) contained: 140 mM NaCl, 5 mM KCl, 8 mM Na2HPO4 at pH 7.3. Phosphate buffered saline with Tween (PBST) consisted of 0.1% Tween 20, 20 mM Na2HPO4, 115 mM NaCl; pH 7.4. The collagenase Solution for perfusion contained 30 mg collagenase type IV in 100 ml PBS solution.
5
Tumor Dissociation and Single-Cell Isolation
6
RNA Extraction and cDNA Synthesis Protocol
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Guanidine thiocyanate—phenol solution (Qiagen), Revert-Aid H Minus First Strand cDNA Synthesis Kit (Fermentas), 2X PCR Master Mix (Fermentas), DNase (Ambion) and veriquest SYBR green qPCR master mix (affymetrix) were purchased through local suppliers. Agarose, Tris base, Glacial acetic acid, EDTA–Na2 and other chemicals were of molecular grade, purchased from E-Merck, India. LB broth, LB Agar, ampicillin, X-Gal and IPTG were purchased from Himedia, India. Hyaluronidase type IV was purchased from Sigma-Aldrich, India. The primers used were synthesized by Integrated DNA Technology, India.
7
Muscle Fiber Transfection in Adult Mice
8
Cumulus Cell Dispersion Assessment
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Crisp1+/− and Crisp1−/− COC were incubated at different times in 100 µl of medium alone or mixed with 0.3 mg/ml hyaluronidase (type IV; Sigma-Aldrich) at 37°C in an atmosphere of 5% CO2 in air, and then observed under a stereoscopic microscope (SMZ800; Nikon). The cumulus dispersion status was classified from 0 to 4, 4 being the dispersion observed in intact COC and 0 in denudated eggs. Intermediate values were assigned for intermediate status.
9
Intramuscular Plasmid Injection and Electroporation
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Intramuscular plasmid injection and electroporation were performed according to Schertzer et al. [54 (link)]. In brief, the animals were anesthetized using 5% isoflurane to inject their footpads with 2 mg/ml hyaluronidase type IV (Sigma-Aldrich Co.) and injected 1 h later with 20 μg Mito-ds-red plasmid (Takara Bio Inc, USA). Electroporation was then performed using acupuncture needles as electrodes and delivering 20 pulses of 100 V and 20-ms duration at 1 Hz using a pulse stimulator (Grass S48; W. Warwick, RI, USA).
10
Plasmid Transfection in Rat FDB Muscles
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Transfection of plasmids in FDB muscles of adult rats was performed as described previously (DiFranco et al., 2009 (link)). In brief, the animals were anesthetized using 5% isoflurane to inject their footpads with 2 mg/ml hyaluronidase type IV (Sigma-Aldrich), and 1 h later, 40–60 µg total cDNA was injected (20–40 µl) followed by electroporation using acupuncture needles as electrodes and delivering 20 pulses of 100 V and 30-ms duration at 1 Hz using a square pulse stimulator (model SD9; Grass Technologies) combined with an oscilloscope (model TDS 220; Tektronix). Mfn1 or Mfn2 silencing was performed by injection of the fluorescent proteins expressing cDNAs, plus 400 pmol of siRNA. In every experiment, NT-siRNA was injected to the left footpad and Mnf1 or Mfn2 siRNA to the right footpad. 7–10 d after electroporation, the animals were euthanized and the FDB muscles were harvested. FDB muscles were digested using 3 mg/ml collagenase type 2 (Worthington Biochemical Corporation) for 2–3 h at 37°C under agitation, followed by mechanical dissociation, adapted from Casas et al. (2010) (link). Isolated fibers were plated onto CELL-TAK–coated (BD) coverslips in DMEM/F12 medium (Lonza) supplemented with penicillin/streptomycin and 10% horse serum (HS), and used within 24 h after plating.
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