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Millex hv pvdf

Manufactured by Merck Group
Sourced in United States

The Millex-HV PVDF is a laboratory filtration product manufactured by Merck Group. It is a high-flow, low-protein-binding syringe filter made from polyvinylidene fluoride (PVDF) material. The core function of the Millex-HV PVDF is to filter samples prior to analysis or further processing.

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11 protocols using millex hv pvdf

1

Lentiviral Vector Production in HEK293T Cells

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The day before transduction HEK293T cells were seeded at a density of 10 million cells per transfer vector in 15 cm plates in a volume of 20 ml of DMEM 10% FBS without penicillin/streptomycin. Each of the transfer vectors, together with packaging plasmids pmDLg/pRRE (#12251) and pRSV-REV (#12253) along with envelope plasmid pMD2.G (#12259), were combined to a total of 12 µg at a ratio of 4:2:1:1, respectively, in 2 ml of serum free DMEM without phenol red.
PEI 1 mg mL−1 was batch tested and added at a ratio of 6:1 PEI: DNA. The solution was briefly vortexed and incubated at room temperature for 15 min. Following this the solution was added dropwise to the cells. Flasks were rocked gently in a circular motion to distribute the precipitates, and then returned to the incubator. Twenty-four hours later fresh DMEM supplemented with 10% FBS and 1% penicillin/streptomycin was added. The viral supernatant was collected 72 h after transduction, cleared by centrifugation at 500 × g for 5 min at 4 °C then passed through a 0.45 µm pore PVDF Millex-HV (Millipore). Lentivirus was aliquoted and stored at −80 °C for future use.
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2

Ibuprofen Tablets Dissolution Analysis

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Dissolution studies were performed with apparatus II (paddle) according to the USP specifications for ibuprofen tablets.10 The dissolution characteristics of at least 12 tablets of each batch were studied at pH 7.2 in phosphate buffer at 37°C ± 0.5°C. The dissolution tests were performed in an Erweka DT80 (Heusenstamm, Germany) at a rotating speed of 50 rpm. Samples (5 mL) were withdrawn at different sampling times (0, 5, 10, 15, 20, 30, 45 and 60 minutes) without reposition. Samples were filtered immediately after sampling with a 0.45 µm filter (hydrophilic PVDF Millex-HV, Millipore, Billerica, MA, USA). Ibuprofen was measured by UV spectrophotometry at 266 nm in a Beckman Coulter Du-6 spectrophotometer (Brea, CA, USA). The initial release rate was estimated as the ibuprofen (% of theoretical dose) released during the first 5 minutes (Q5). This parameter was used to compare differences on the release between the formulations.
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3

Lentiviral Particle Generation Protocol

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Lentiviral particles for shNTC and shStau2 were generated from HEK-293T cells cotransfected with psPAX2, pcDNA3.1-VSV-G and the respective pFu3a plasmids using calcium phosphate coprecipitation. After 48 h virus production, supernatants were filtered (0.45 µm PVDF Millex-HV; Millipore, Burlington, MA, USA), concentrated by ultracentrifugation (65,000× g, 140 min, SW 32 Ti rotor; Beckman Coulter, Brea, CA, USA) and resuspended in Opti-MEM™ (Life Technologies, Carlsbad, CA, USA) [19 (link)].
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4

Lentiviral Particle Production Protocol

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Lentiviral particles for shNTC, shStau2, shPum2, shAgo2, shAgo1 and eGFP-Ago2 were generated from HEK-293T cells co-transfected with packaging plasmids psPAX2 and pcDNA3.1-VSV-G and the respective lentiviral Fu3a plasmid using calcium phosphate coprecipitation. After 48 h virus production, supernatants were filtered (0.45 μm PVDF Millex-HV; Millipore), concentrated by ultracentrifugation (65 000 × g, 140 min, SW 32 Ti rotor; Beckman Coulter) and resuspended in Opti-MEM™ (Life Technologies) (16 (link)).
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5

Lentiviral Particle Production Protocol

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Lentiviral particles for shNTC, shHuR, shAgo2, miR-scr, miR-26a, eGFP-Stop and eGFP-miR-26-sponge were generated from HEK-293 T cells co-transfected with psPAX2, pcDNA3.1-VSV-G and the respective pFu3a plasmids using calcium phosphate coprecipitation. After 48 h virus production, supernatants were filtered (0.45 µm PVDF Millex-HV; Millipore), concentrated by ultracentrifugation (65,000xg, 140 min, SW 32 Ti rotor; Beckman Coulter) and resuspended in Opti-MEM™ (Life Technologies) [12 (link)].
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6

Standardized pH Measurement Protocol

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pH measurements were carried out using Mettler Toledo Seven Easy pH meter (model S20) which was routinely calibrated with standard buffers. The experiments were performed at the 20 mM pH 7.4 sodium phosphate buffer. All preparations used in the experiments were filtered through 0.45 μm Millipore Millex-HV PVDF filter.
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7

Nasal Lining Fluid Proteomic Analysis

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We used 7.5 ml of saline for washing each nasal cavity, which totalled 15 ml of saline per person. The collected fluid samples were combined and centrifuged at 500g x to separate the cells for cell counting and cell type analysis. The supernatant was centrifuged for the second time at 4000g x, before it was filtered through the 0.45 μm membrane (Millex-hv PVDF, Millipore) and divided into aliquots. Samples were frozen to -70°C for further use. Protein concentrations (RC DC Protein Assay, BioRad) of the NFL samples varied between 0.03 and 0.19 mg/ml. A total of 7–9 ml of each NLF sample was concentrated with ultrafiltration (Amicon Ultra-15 5000 MWCO, Millipore, Ireland) to 250 μl which was used in the proteomic analysis. For immunoblotting, 12 μl of untreated NFL was loaded onto the gel lane and for ELISA 250 μl was evaporated in SpeedVac to 50 μl.
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8

HPLC Analysis of Chemical Compounds

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A .45 μm Millex-HV PVDF (Millipore, New Bedford, MA, USA) membrane was used to
filter the sample. Using an analytical column (Phenomenex® ODS 100 A 250 mm ×
4.60 mm, 5 μm) accompanied by a C18 guard column (2.0 cm 4.0 mm; 5 m), HPLC
analysis was carried out on a Shimadzu chromatographer outfitted with a ternary
pump (Shimadzu LC-20AT) and diode assay detector (Shimadzu SPD-M20A, Kyoto,
Japan). Data processing was carried out using LC solutions software (version
1.25). Acetonitrile and water were used as the mobile phase in a gradient
chromatography procedure, with the initial acetonitrile/water ratio being 2:8
v/v and the final acetonitrile/water ratio being 8:2 v/v at 1 mL/min flow rate
over the course of 30 min with 25oC column temperature and 20 μL
injection volume. Every day, the mobile phase was prepared and sonicated to
remove gas before usage. The UV spectra were observed between 450 and 200 nm.
All of the standard solutions and extracts were made with ethanol. The
concentration of the solution chemical was 2000 g/mL, whereas the reference
solutions were employed in 6 different concentrations (12.5, 25.0, 100.0, 150.0,
250.0, and 500.0 g/mL).22
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9

HIV-eGFP Viral Infection Assay

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The HIV-1 vector and plasmid used in this study have been described previously59 (link). HIV-eGFP virus (in which nef was replaced with the eGFP reporter gene) was produced by cotransfection with a ratio of 1:7 of pMD2.G together with pNL4–3_GFP_R-Env-60 (link) (both plasmids were obtained from the NIH AIDS Research and Reference Program, Division of AIDS, NIAID, NIH, Bethesda, Maryland, USA). Vector containing supernatants were harvested 48 h after transfection in 293 T cells, cleared by centrifugation, filtered by a 0.45 mm filter (MILLEX-HV PVDF; Millipore, Carrigtwohill, County Cork, Ireland), and stored at −80 °C.
CTR, M1-MDM and M12 MDM were infected with 60 µl/well of HIV-eGFP viral stock. Infected MDM (5 × 105 cells/condition) were detached from plastic adhesion after incubation with Accutase (150 µl/well for 30 min at 37 °C), as described46 (link), spun, and their pellet was resuspended in a fixing solution containing 4% paraformaldehyde (PFA). Flow cytometry for GFP expression was performed using a FACS Calibur instrument (Becton Dickinson Italia, SpA, Milano, Italy), and the results were analyzed with the FlowJo software version 8.4.3 (Tree Star, Ashland, Oregon, USA).
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10

Protein Concentration Determination

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Protein stock was prepared in 20 mM sodium phosphate buffer pH 7.0, and concentration was measured by using the extinction coefficient at E280nm1%
 = 12.0 on Perkin Elmer (Lambda 25) double beam spectrophotometer attached with peltier temperature programmer (PTP-1). A filtered buffer through a 0.45 μm Millipore Millex-HV PVDF filter was used throughout the study.
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