The transgene expression cassette included AAV2 terminal repeats, the hybrid cytomegalovirus/chicken beta-actin promoter, human wild-type TDP43, and the bovine growth hormone polyadenylation sequence76 (link). Helper and AAV9 capsid plasmids were from the University of Pennsylvania77 (link). The expression cassette was packaged into recombinant AAV9 as previously described76 (link). Viral stocks were sterilized using a Millex-GV syringe filter (Millipore), aliquoted, and frozen. Viral genome copies were titered using a dot-blot assay.
Millex gv syringe filter
Manufactured by Merck Group
Sourced in United States, Ireland
The Millex-GV syringe filter is a laboratory filtration device. It is designed to remove particulates from liquid samples during the sample preparation process.
Automatically generated - may contain errors
Lab products found in correlation
Lactated ringer s solution, by Baxter (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Superblock, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose filter, by Merck Group (1 mentions)
Dneasy tissue kit, by Qiagen (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
Synergy 2 microplate reader, by Agilent Technologies (1 mentions)
Dmem low glucose, by Nacalai Tesque (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Penicillin streptomycin amphotericin b suspension, by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Minispin plus, by Eppendorf (1 mentions)
Bx 63 upright fluorescent microscope, by Olympus (1 mentions)
Cm dii, by Thermo Fisher Scientific (1 mentions)
Permeabilization buffer, by BD (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Gemini nx c18 5 m column, by Phenomenex (1 mentions)
23 protocols using millex gv syringe filter
1
AAV9-Mediated Expression of TDP43
2
Seawater Sampling and DNA Extraction
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A volume of 800–1,000 mL seawater was collected at the bottom of the tanks using a silicon hose fixed at the edge of the tanks to prevent contamination from the upper surface layer of oil at each sampling time (day 1, 2, 7, 14, and 28). Samples were immediately filtered through nitrocellulose filters (pore size, 0.22 μm) (Millipore, Billerica, MA, USA) and the filters were stored at −80°C until DNA extraction. DNA extraction was performed using the protocol described by Preston et al. (45 (link)) with the following modifications: the total volume of the lysate was used after filtration through a Millex-GV syringe filter (pore size, 0.2 μm; diameter, 13 mm) (Millipore) and a proportionally higher volume of diluent (555 mM sodium acetate pH 5.2 in 70% ethanol [v/v]) was added. This was then passed through a column in the DNeasy Tissue Kit (Qiagen, Hilden, Germany) and washed twice before DNA was eluted with 80 μL elution buffer.
3
AAV9-Mediated Gene Delivery in CNS
4
Exosome-mediated T-cell activation by bispecific antibodies
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MB49-EpCAM-OVA cells were heat-shocked at 45°C for 10 min to induce necrosis, followed by incubation at 37°C overnight. Alternatively, MB49-EpCAM-OVA were cultured to 100% confluency, cell culture media was collected and exosomes were isolated using Total Exosome Isolation kit (Invivogen). Exosomes were further purified by filtering through an Amicon Ultra-15 Centrifugal Filter (Millipore) and a 0.22 µm Millex-GV syringe filter (Millipore). The purity of exosomes was analyzed using Uncle (Unchained Labs), and exosome yield was quantified using a BCA Protein Assay (Pierce). hCD40tg DCs were co-cultured with CellTrace Violet-labeled OT-1 T cells and necrotic debris or exosomes from MB49-EpCAM-OVA cells in the presence of 5 nM or 100 nM CD40×EpCAM bsAb, respectively, or control antibodies for 72 hours. OT-1 T-cell proliferation was assessed using an FACSVerse.
5
Cyanophage Propagation and Enumeration
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Three T7-like cyanophage strains that infect Prochlorococcus MED4 were used in this study: P-SSP7, P-GSP1 and P-TIP38. Their ability to infect the resistant Prochlorococcus strains is shown in Supplementary Table S1 . Phages were propagated from single plaques in liquid cultures of exponentially growing host cells. Cell debris were removed from viral lysates by centrifugation (10 000 g at 20 °C for 15 min) and filtration (0.22 μm Millex GV syringe filter, Millipore (Cork, Ireland); or 0.2 μm Rapidflow filter unit, Nalgene, Rochester, NY, USA). Phages were enumerated by plaque assays in which serially diluted phages were plated in pour plates after a 1–4 h adsorption step to host cells in liquid under host growth conditions.
6
Iterative Evolution of Cyanobacterial Phages
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Initially, aliquots of the ancestral populations were used to infect 1 ml liquid cultures of exponentially growing host cells in 48-well microplates. S-TIM4 was used to infect five bacterial cultures of each of the hosts: Synechococcus sp. WH8102, Prochlorococcus sp. MED4, and Prochlorococcus sp. MIT9515. Syn19 was used to infect five bacterial cultures of the Synechococcus WH8102 host. Cyanobacterial cultures were monitored by measuring chlorophyll a fluorescence using a Synergy2 Microplate Reader (Ex/Em: 440/680 nm; BioTek) which is a proxy for cell density51 (link). Once the reduction in the cell densities of infected cultures ceased (while growth of the uninfected control continued), viral lysates were filtered (0.22 μm Millex GV syringe filter, Millipore (Cork, Ireland)). This enabled us to avoid the possibility of transferring resistant cells, and as a result, allowing coevolutionary dynamics. Filtered lysates were stored in glass bottles at 4 °C. To start a new lysis cycle, a fixed volume of 50 μl of the filtered lysate was used to infect 1 ml of the naïve cyanobacterial hosts of the same strain each lysate grew on. Overall, 15 cycles were conducted, the length of individual infection cycles ranged between ~1 and 3.5 weeks.
7
Production of AAV8-PCSK9 Viral Particles
8
AAV Vector Production and Characterization
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The transgene cassette was composed of AAV serotype 2 inverted terminal repeats, the hybrid cytomegalovirus/chicken β-actin promoter, a GFP construct or TDP-43 construct, the woodchuck hepatitis virus posttranscriptional regulatory element, and the bovine growth hormone polyadenylation sequence.28 (link) This cassette was packaged into AAV1, AAV8, AAV9, or AAV10 using helper and capsid plasmids from the University of Pennsylvania.1 (link),29 (link) Viral stocks were sterilized via Millipore (Billerica, MA) Millex-GV syringe filter, aliquoted, and frozen. Stocks were titered by dot blot assay, and equivalent viral titers were created by diluting in lactated Ringer’s solution (Baxter Healthcare, Deerfield, IL). Through the course of these studies, we made and used three batches of AAV1 GFP, one batch of AAV8 GFP, seven batches of AAV9 GFP, two batches of AAV10 GFP, and one batch of AAV9 TDP-43. Consistent results across vector batches were found in the cases where more than one preparation was used.
9
Packaging of PCSK9 into AAV8 Virus
10
AAV9 Vector Cassette Characterization
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The vector cassette was composed of AAV serotype 2 inverted terminal repeats, the hybrid cytomegalovirus/chicken β-actin promotor, a GFP, FUS, or TDP-43 construct, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and the bovine growth hormone polyadenylation sequence [52 (link)]. The vector cassette was packaged into AAV9 using helper and capsid plasmids from the University of Pennsylvania [53 (link), 54 (link)]. All vectors were prepared under the same laboratory conditions. Viral stocks were sterilized via Millipore Millex-GV syringe filter (Billerica, MA), titered by dot blot assay, aliquoted, and stored frozen until use.
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