Mice were treated with 30% HPCD (catalog no.: 332607; Sigma-Aldrich, St Louis, MO) from 12 to 14 months of age. This solution was prepared by diluting under the hood the 45% HPCD stock in sterile water for injections (catalog no.: 46066-808-25; Aspen Veterinary Resources, Liberty, MO). The dilution was with phosphate-buffered saline sterilized by 0.22 μm filtration (SCGP00525; EMD Millipore Corporation, Billerica, MA). HPCD was usually administered in the morning by subcutaneous injections at a 2 g/kg of body weight dose twice a week for 2 months, the treatment paradigm chosen based on our previous study (25 (link)). Control animals received injections with similar amounts of diluted phosphate-buffered saline. No indication of swelling, redness, irritation at the site of injection or of whole-body toxicity was evident for the treatment duration as assessed by lack of mortality or signs of physical distress, such as hunched posture, lethargy, fur ruffling, or respiratory distress. Of pertinence is that a similar HPCD dosing in human subjects with Niemann-Pick disease type C1, except that HPCD was delivered intravenously or intrathecally, raised no safety concerns (20 (link)).
Scgp00525
Manufactured by Merck Group
Sourced in United States
SCGP00525 is a piece of laboratory equipment manufactured by Merck Group. It is designed for general laboratory use, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
P3932, by Merck Group (2 mentions)
Magnetic luminex performance assay human highsensitivity cytokine base kit, by R&D Systems (2 mentions)
Bio plex manager 5, by Bio-Rad (2 mentions)
Bio plex 200, by Bio-Rad (2 mentions)
Ufc900324, by Merck Group (2 mentions)
Hisprep ff 16 10, by GE Healthcare (2 mentions)
Kta explorer 10, by GE Healthcare (2 mentions)
Kta purifier 100, by GE Healthcare (2 mentions)
Vivaspin 20, by Sartorius (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mirneasy mini spin columns, by Qiagen (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Surgical scissors, by Fine Science Tools (1 mentions)
Pierce lal chromogenic endotoxin quantification kit, by Thermo Fisher Scientific (1 mentions)
Hiprep 26 10 desalting column, by GE Healthcare (1 mentions)
V3021, by Promega (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Ultracentrifuge, by Beckman Coulter (1 mentions)
Nanodrop assay, by Thermo Fisher Scientific (1 mentions)
21 protocols using scgp00525
1
Cyclodextrin Treatment in Mice
2
Poly(2-HEMA) Coated Cell Encapsulation
3
Cytokine Quantification in iAEC2s
4
Mouse Aortic VSMC Isolation Protocol
5
Quantifying Staphylococcus aureus Virulence
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This assay was adapted from a previously published protocol (Pang et al., 2010 (link)). We added a 1:10 dilution of E. coli CFCM containing AIP-1 into 1.5 ml cultures containing 1:100 inoculums of either wild-type or agrA-deficient S. aureus JE2. A separate set of tubes was prepared identically plus the addition of a 1:2 dilution of 3 kDa-filtered C. striatum CFCM. These cultures were incubated at 37°C shaking at 200 RPM for 4 h. After incubation, CFCM was generated from each S. aureus culture by passage through a 0.2 μm filter (Millipore, #SCGP00525). Rabbit blood (Hemostat Laboratories, USA) was diluted v/v to 3% in sterile PBS then 70 μl was combined with 30 μl of each S. aureus CFCM in eight replicates for each condition in a 96-well plate. Blood was also combined at the same ratio with BHI as a negative control and BHI was used as a blank for OD630 measurements. Plates were incubated at 37°C for 40 m then OD630 was read to determine rabbit erythrocyte lysis.
6
Production and Purification of Quorum Sensing Autoinducer Peptide
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AIP-1 production was performed similarly to methods described previously (Thoendel and Horswill, 2009 (link)). Briefly, E. coli strain AH594 carrying an arabinose-inducible AIP-1 synthesizing gene locus (PBAD-agrBD) was grown overnight at 37°C shaking at 200 RPM in 5 ml of LB with 100 μg/ml ampicillin. A 1:50 dilution of overnight culture was added to 50 ml LB without ampicillin in a 250 ml flask and was shaken at 200 RPM incubated at 37°C for 90 min prior to addition of arabinose to a final concentration of 0.02%. The induced culture was further incubated in the same conditions for 4 h. Next the cell culture was transferred to a sterile 50 ml conical centrifuge tube and cell supernatants were removed after centrifugation at 7,000 g for 10 m at 4°C. Cell supernatants were next passed through a 0.2 μm filter (Millipore, #SCGP00525) to remove remaining cell debris and then passed through a 3 kDa-cutoff size exclusion filter (Millipore #UFC900324). Filtrates were prepared for storage by addition of sterile glycerol and dithiothreitol to concentrations of 10% and 1 mM respectively. Aliquots of 1.5 ml each were stored at -80°C and exhibited full activity for >30 days.
7
Histidine-Tagged DCN Purification
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The IMAC purification of human DCN tagged with histidine/asparagine was performed with a HisPrep FF 16/10 affinity chromatography column (GE Healthcare, USA), controlled by the FPLC system Äkta Explorer 10 (GE Healthcare). DCN elution was identified by an increased absorption at 280 nm and 256 nm. All DCN samples were pooled and desalted with a HiPrep™ 26/10 desalting column (GE Healthcare), controlled by the Äkta Purifier 100 (GE Healthcare). After washing and concentrating with ultrafiltration units (Vivaspin 20, Sartorius, Germany), the DCN samples were sterile filtered (SCGP00525, Millipore, USA) and subsequently stored at −80 °C. All DCN samples were tested for endotoxin contamination by the Pierce LAL chromogenic endotoxin quantification kit (Thermo Scientific GmbH, Schwerte, Germany) with E. coli LPS as a standard. The level for 50 µg/mL DCN was 1.2 ± 0.31 EU/mL.
8
Cytokine Quantification in iAEC2s
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Supernatants of corrected and mutant iAEC2s (n = 3 independent wells) were filtered through 0.22 μm filters (Millipore SCGP00525) and analyzed using a customized Magnetic Luminex® Performance Assay Human HighSensitivity Cytokine Base Kit (R&D Systems, Inc). Mean fluorescence intensity was measured to calculate final concentration in pg/ml using Bioplex200 and Bioplex Manager 5 software (Bio-Rad) and adjusted to cell number.
9
Corynebacterium Cell-free Conditioned Medium
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C ell-f ree c onditioned m edium was generated from each indicated Corynebacterium spp. after inoculation into BHI cultures and growth at 37°C shaking at 200 RPM for 48 h. Conditioned medium was removed after centrifugation of 48-h cultures for 10 m at 7,000 g and then passed through a 0.2 μm filter (Millipore, #SCGP00525) yielding CFCM, which was either used directly for experiments, frozen at -80°C prior to use or further passed through a 3 kDa molecular weight size exclusion filter (Millipore #UFC900324) then frozen at -80°C. For some samples, CFCM was heat treated at 95°C for 15 m. For other samples, CFCM was Proteinase K (Promega #V3021) treated by addition of 100 μg in 1 ml of CFCM prior to incubation at 55°C for 1 h followed by a 95°C heat inactivation for 10 m prior to testing for the effect on AIP-1.
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10
Isolation and Purification of Small Extracellular Vesicles
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A classical differential UC protocol (31 (link)) was used with minor modifications to isolate sEVs. 5 × 106 HPSC/HPaStec cells were seeded in a T150 cm2 flasks (corning) at 50% confluence in each respective medium with 10% exosome-free FBS (Thermo Scientific, #A2720803), and the conditioned medium was harvested when cells were 70% to 80% confluent (48 h). Cell-conditioned media was cleared of cells, cell debris, and large membrane vesicles by sequential centrifugation at 500g for 30 min followed by 12,000g for an additional 30 min and was then filtered through a 0.22 μM filter unit (Millipore, #SCGP00525). This step was done to maximize quality over quantity to exclude the apoptotic bodies and other debris supported by previous publications (24 (link), 28 (link), 32 (link)).To sort by density, sEVs were collected from the cleared supernatants after centrifugation at 100,000g for 2 h in SWT32i (Beckman) swinging buckets using a Beckman Coulter ultracentrifuge (Beckman). The sEVs were washed with PBS and repurified by centrifugation at 100,000g for 2 h. The pellets were resuspended in 0.22 μM–filtered PBS. Exosomal protein quantity was estimated by Pierce bicinchoninic acid (BCA) protein assay reagent (Thermo Scientific, #23227) and Nanodrop assay (Thermo Scientific), according to the manufacturer’s instructions.
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