One- to two-week old pre-weaned Yorkshire pig islets were isolated under procedures approved by the University of California Irvine, Institutional Animal Care and Use Committee. Pancreata were procured and stored in cold HBSS (<1 hour). Pancreata were minced and digested with Collagenase Type V (Sigma-Aldrich) in a 37°C shaking water bath for 15 minutes. The enzymatic digestion was quenched with HBSS supplemented with 1% porcine serum (Gibco-Thermo Fisher Scientific) and digested tissues were filtered through a 500 μm metal mesh. Islets were then cultured in maturation media made up of Ham’s F-12 medium (Corning), HEPES (Sigma-Aldrich), L-glutathione (Sigma-Aldrich), nicotinamide (Sigma-Aldrich), ITS+3 (Sigma-Aldrich), gentamycin sulfate (Corning), Trolox (Sigma-Aldrich), heparin (Sagent Pharmaceuticals), Pefabloc (Santa Cruz Biotechnology), L-glutamine(Alfa Aesar), medium 199 (Corning) and 10% porcine serum.
Porcine serum
Manufactured by Thermo Fisher Scientific
Sourced in United States, New Zealand, Germany
Porcine serum is a biological fluid derived from the blood of pigs. It contains a variety of proteins, growth factors, and other biomolecules that can be used in various cell culture and research applications.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (2 mentions)
Gentamycin sulfate, by Corning (1 mentions)
Collagenase type 5, by Merck Group (1 mentions)
Pefabloc, by Santa Cruz Biotechnology (1 mentions)
Medium 199, by Corning (1 mentions)
Its 3, by Merck Group (1 mentions)
L glutathione, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Trolox, by Merck Group (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
Ham s f 12 medium, by Corning (1 mentions)
Sigma type 5 colla genase, by Merck Group (1 mentions)
Mannose receptor, by Abcam (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Nucblue fixed cell stain, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Casein blocking buffer, by Merck Group (1 mentions)
Spectrophotometer epoch microplate reader, by Agilent Technologies (1 mentions)
7 protocols using porcine serum
1
Isolation and Culture of Porcine Islets
2
Isolation of Porcine Pancreatic Islet Progenitor Cells
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PPIs were isolated from pancreata of 8–15-day-old, pre-weaned Yorkshire piglets as previously described [44 (link)]. Briefly, pancreata were procured in less than 10 min and stored into cold HBSS (cold ischemic time < 1 h). Pancreata were minced into 1 mm3 pieces and digested with Sigma Type V Colla-genase (cat#C8051, Sigma Aldrich, Buchs, Switzerland) dissolved in HBSS (2.5 mg/mL) in 37 °C, 100 rpm shaking water bath for 15 min. After quenching with HBSS supplemented with 1% porcine serum (cat#26250084, Gibco, Waltham, MA, USA), digested tissues were filtered through a 500 μm metal mesh.
3
Immunofluorescence Staining of Macrophages
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The CD14+ cells (1 × 106) were cultured on slides for 7 days at 37°C in a humidified atmosphere containing 5% CO2 to obtain MDM, and the cells were washed in PBS and fixed in 2% formaldehyde for 15 min. To avoid unspecific binding, the MDM were blocked for 30 min with 2% porcine serum (Gibco) in PBS (PBS-PS). Posteriorly, the cells were incubated 1 h with primary mAbs to Mannose Receptor (4 μg/mL, Abcam) TCRαβ (4 μg/mL, Thermo Fischer) and CD3 (1:100 cell signaling). The following were used as secondary mAbs (diluted in PBS-PS): donkey anti-rabbit IgG Alexa Fluor-488 (1:100) and donkey anti-rat IgG Alexa Fluor-647 (1:100) provided by Jackson ImmunoReseach and Goat anti-Mouse IgG Alexa Fluor-546 provided by Invitrogen, cells were incubated 1 h. Finally, the samples were washed and incubated with 4_,6-diamidino-2 phenylindole dihydrochloride (DAPI; NucBlue Fixed Cell Stain, Molecular Probes) for 10 min for nuclei labeling and mounting with ProLong Gold Antifade Mountant (Invitrogen). The slides were examined by confocal microscopy FV-1,000 Olympus, and FIJI software was used for the analysis.
4
Measurement Conditions in Biological Buffers
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All measurements were conducted in PBS buffer, Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, Buchs, CH) solubilized in PBS buffer (pH 6.2), or artificial wound exudate (Campbell et. al (2003) with following modification: AWE = DMEM solubilized in PBS buffer (pH 6.2), including 10% (v/v) porcine serum (Gibco, Lucerne, CH)).
5
Competitive ELISA for Nipah Virus Detection
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Nunc 96–Well non–surface treated Microplates (Thermo Fisher Scientific, MA, USA) were coated with 100 μl (12 ng/well) of the recombinant NiV–G in 0.06 M carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C. After washing 5 times with PBS–T, plates were blocked with 5% commercial porcine serum (Life Technologies, New Zealand) in Casein blocking buffer (Sigma–Aldrich, SL, USA). A commercial normal pig serum was used as a negative control, and one serum collected at 28 dpi from NiV–B inoculated pig (#7) was used as a positive control in the cELISA. Negative and positive serum controls are included in each ELISA plate to calculate percent inhibition (PI) results for test samples. Thus after blocking, test serum samples, positive, and negative serum controls (50 μl/well, 1:10 in PBS–T) were added in duplicate, then an equal volume of the hybridoma culture supernatant (F20NiV−65, 1:500 in Casein blocking buffer) were added at the same time. Following 1 h incubation and washing, the HRP–conjugated anti–mouse IgG (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added. Then 3,3′,5,5′–Tetramethylbenzidine (TMB, Pierce Biotechnology Inc. IL, USA) was added. The reaction was stopped using 2N Sulfuric acid and the OD was determined at 450 nm using a BioTek Epoch Microplate Spectrophotometer reader. Results of PI were calculated based on the following formula:
6
Porcine Islet Transplantation Methodology
7
Optimized Isolation and Culture of Primary Murine Brain Endothelial Cells
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Cell culture flasks, plates, and other plasticware were purchased from Greiner Bio-One (Kremsmünster, Austria). Cell culture medium M199 and MCDB131, minimal essential medium, DMEM/F-12 (Ham's), porcine serum, and dispase were purchased from Life technologies; collagen G from bovine calf skin was obtained from Biochrom (Berlin, Germany), and cell culture additives were from PAA laboratories (Pasching, Austria).
Collagenase/dispase was from Roche Applied Science. Transwell multiwell plates (polyester membrane inserts, 0.4 µm pore size) as well as simvastatin, cholesterol, hydrocortisone, Percoll (pH 8.5-9.5) and Aβ Johannes-Gutenberg-University Mainz, Germany. For immunoblot analysis of mBCEC, primary antibodies were purchased from Sigma-Aldrich (Anti-Amyloid Precursor Protein, C-Terminal, Cat#A8717) and from Santa Cruz Biotechnology (clusterin/apoJ (H-330), Cat# sc-8354). Simvastatin used for animal studies was purchased from MSD Austria. PCR reagents were from Bio-Rad and primers were purchased from Invitrogen. Pre-validated primers were purchased from Qiagen (QuantiTect Primer Assay). All other reagents and chemicals were purchased either from Sigma-Aldrich or Merck.
Collagenase/dispase was from Roche Applied Science. Transwell multiwell plates (polyester membrane inserts, 0.4 µm pore size) as well as simvastatin, cholesterol, hydrocortisone, Percoll (pH 8.5-9.5) and Aβ Johannes-Gutenberg-University Mainz, Germany. For immunoblot analysis of mBCEC, primary antibodies were purchased from Sigma-Aldrich (Anti-Amyloid Precursor Protein, C-Terminal, Cat#A8717) and from Santa Cruz Biotechnology (clusterin/apoJ (H-330), Cat# sc-8354). Simvastatin used for animal studies was purchased from MSD Austria. PCR reagents were from Bio-Rad and primers were purchased from Invitrogen. Pre-validated primers were purchased from Qiagen (QuantiTect Primer Assay). All other reagents and chemicals were purchased either from Sigma-Aldrich or Merck.
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