293T cells were seeded in 10 cm plates and grown to 70-80% confluency before cell transfection. The lentiviral transfer vector plasmid, packaging plasmid (psPAX2), and envelop plasmid (pMD2.G) were combined at 4:3:1 ratio (12.5 μg:9.375 μg:3.125 μg, respectively) in 1 mL of 0.25 M CaCl2. The mix of plasmid DNA was added into 1 mL of filtered 2× concentrate BES buffered saline (Millipore 14280, bioWORLD 40220006-2) in a dropwise manner. A sterile 1 mL pipette was used to gently create bubble air through the DNA mix. Following the incubation at room temperature for 5–10 mins. the solution was added dropwise to 293 T cells. The plates were rocked gently in a circular motion to distribute the precipitates. Cells were cultured at 37 °C in a humidified 5% CO2 incubator for 16 hours, followed by replacement with fresh media. Viral supernatants were collected twice at 24 hours and 48 hours after replenishing new media. The supernatant was passed through a 0.45 μm pore PVDF Millex-HV filter (Millipore, SLHVR33RB) and stored at −80 °C.
Pvdf millex hv filter
Manufactured by Merck Group
The PVDF Millex-HV filter is a laboratory filtration device designed for high-volume filtration. It is constructed with a polyvinylidene fluoride (PVDF) membrane, providing a reliable and efficient filtration solution for various laboratory applications.
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4 protocols using pvdf millex hv filter
1
Lentiviral Vector Production in 293T Cells
2
Lentiviral Vector Production in HEK293T Cells
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HEK293T cells were cultured in 15-cm plates and grown to 90% confluency before cell transfection. The lentiviral transfer vector DNA, together with psPAX2 packaging and pMD2.G envelope plasmid DNA was combined in a ratio of 4:3:1, respectively. A sterile 1-mL pipette was used to vigorously bubble air through the DNA mix, during which 1.5 mL 2× N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)–buffered saline (280 mM NaCl, 50 mM HEPES, 1.5 mM Na2HPO4, and pH 7.0) was added dropwise into the precipitate. After the incubation of the solution at room temperature for 5 minutes, the solution was added dropwise to HEK293T cells. The plates were rocked gently in a circular motion to distribute the precipitates and then returned to the incubator at 5% CO2. Cells were replaced with fresh growth medium which was added 24 hours after transfection. The viral collection was performed at 48 and 72 hours after the transduction. The supernatants from the 2 harvests were passed through a 0.45 μm pore PVDF Millex-HV filter (Millipore) and stored in the freezer at −80 °C for subsequent uses.
3
Lentiviral Transduction of Mouse Xenograft Cells
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1 × 106 HEK293T cells were seeded on 10-cm culture dishes and incubated for 24 h. The gfp-luc2 lentiviral transfer vector with packaging and envelope plasmids (pMDL, RSV-REV and VSVg) were combined at a ratio of 4:2:1:1, respectively, and mixed with Lipofectamine 2000 for transfection according to the manufacturer’s protocol. The viral supernatant was collected at 24, 48 and 72 h post-transfection. Cellular debris was pelleted by centrifugation at 1500 rpm for 5 min at 4 °C followed by filtration using PVDF Millex-HV filter, 0.45 μm (Millipore #SLHV033RS). The filtered lentivirus supernatant was then concentrated using Lenti-X Concentrator (Clontech #PT4421–2) according to the manufacturer’s instruction. For cell transduction, the mouse cell-depleted xenograft cells were seeded on a 10-cm culture dish and transduced with the concentrated lentivirus at MOI 2.0 in the presence of 10 μg/mL Polybrene for 24 h. GFP positive cells were selected using a FACSAria SORP (BD Biosciences, USA).
4
Lentiviral Vector Production in HEK 293T Cells
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HEK 293T cells (5 × 106) were cultured in a 6 cm culture plate to reach a 70–80% confluency. Afterward, 4 h before the transfection, the media was replaced with a serum-free DMEM. A second-generation lentiviral vector system was used in this study, in which the backbone EGFP encoding plasmid (pWPXLd), alongside plasmids for packaging (psPAX2) and VSV-G envelope (pMD2.G), were mixed in a 4:4:1 ratio. To prepare the transfection reagent (TR), 15 μL of PolyFect transfection reagent (Qiagen) was mixed with 170 μL serum-free DMEM. The mixture of plasmids and TR solution was incubated at room temperature for 20 min and then added dropwise to the HEK 293T cells. The culture plate was returned to the incubator (37 °C, 5% CO2). After 16 h, the media was changed with a DMEM containing 5% FBS. Seventy-two hours post-transfection, the supernatant containing viral particles was collected (Fig. 1 ). To remove debris, it was centrifuged (1500 rpm, 10 min, 4 °C), and filtered using a 0.45 μm pore PVDF Millex-HV filter (Millipore). The presence of LV particles in the viral stock was assessed by an ELISA-p24 kit (Pishtaz Teb).
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