To generate VLPs, suspension cultures of Sf9 (2 × 106 cells/mL) were infected with the recombinant baculoviruses Bac-EV71-P1/3CD, Bac-CVA16-P1/3CD, Bac-CVA10-P1/3CD, or Bac-CVA6-P1/3CD at a multiplicity of infection of 1 followed by culturing at 27 °C for 3 days. Sf9 cells from each culture were then collected by centrifugation and lysed with 0.15 M PBS containing 1% NP-40. Cell lysates were centrifuged at 12,000 rpm for 15 min to remove cellular debris, and the resultant supernatants were precipitated overnight at 4 °C with 8% (w/v) polyethylene glycol 8000 and 200 mM NaCl. After centrifugation at 12,000 rpm for 15 min, the resulting pellets were collected and resuspended in 0.15 M PBS buffer, followed by clarification by centrifugation. Next, 20% sucrose cushion and 10–50% sucrose-gradient ultracentrifugation steps were carried out as previously described37 (link). Finally, VLP-rich fractions were pooled and buffer-exchanged into 0.15 M PBS buffer using Amicon Ultra 100 K centrifugal filters (Millipore, USA). For comparison, the control antigen was generated from uninfected Sf9 cells following the same protocol. Purified VLPs and control antigen were quantified using the Bradford protein assay kit (Bio-Rad, USA) according to the manufacturer’s instructions.
Amicon ultra 100 k centrifugal filters
Manufactured by Merck Group
Sourced in United States
The Amicon Ultra 100 K centrifugal filters are a type of laboratory equipment used for the concentration and purification of macromolecules, such as proteins and nucleic acids, through the process of ultrafiltration. These filters feature a molecular weight cut-off of 100 kilodaltons, allowing for the selective retention of larger molecules while smaller molecules pass through the membrane.
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Lab products found in correlation
Rnaimax, by Thermo Fisher Scientific (3 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
X tremegene hp transfection reagent, by Roche (1 mentions)
Vp sf media, by Thermo Fisher Scientific (1 mentions)
Block it alexa fluor red fluorescent control, by Thermo Fisher Scientific (1 mentions)
Infinite pro plate reader, by Tecan (1 mentions)
Opti memtm medium, by Thermo Fisher Scientific (1 mentions)
Exosome spin column, by Thermo Fisher Scientific (1 mentions)
Ab30871, by Abcam (1 mentions)
Ab59479, by Abcam (1 mentions)
Ab65230, by Abcam (1 mentions)
5 protocols using amicon ultra 100 k centrifugal filters
1
Production of Virus-Like Particles
2
Lentiviral Transduction of HBL1 Cells
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For lentiviral infection of HBL1 cells with the construct for CARMA1 L225LI HEK293T cells were transfected each with pMD2.G and psPAX2 lentiviral packaging plasmids using X-tremeGENE HP Transfection Reagent (Roche). As a transfer vector we used 2 μg of a pHAGE construct containing hΔCD2 followed by the T2A and CARMA1 L225LI sequence. After 3 days the virus was concentrated in Amicon ultra 100K Centrifugal filters (Millipore). 200 000 HBL1 cells were transduced via spin infection for 1 h and 720 g at room temperature. For increased efficiency of infection we added Polybrene to a concentratin of 8 μg/ml followed by incubation overnight. Infection rates of HBL1 cells were monitored by anti hΔCD2-APC (eBioscience) staining in flow cytometry.
3
Purification of Recombinant Viral Particles
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Subconfluent C7/10 cells in 150-mm dishes were infected with EIL/VEEV or other chimeric viruses, containing nLuc or GFP genes with varying 5’UTRs under control of an additional SG promoter, at an MOI of 10 PFU/cell for 1 h at 30°C. Then cells were incubated overnight in complete media and then in serum-free VP-SF media (Invitrogen) for 24 to 30 h. These media were harvested before cytopathic effect (CPE) developed and additionally clarified by low-speed centrifugation. Viral particles were then concentrated using Amicon Ultra 100K centrifugal filters (Millipore) as described elsewhere [68 (link)]. In most experiments, viruses were used without further purification. In some experiments, concentrated samples were purified further by ultracentrifugation in continuous 20–50% sucrose gradients in a SW-40 rotor at 38,000 rpm for 3 h at 4°C. Visible bands of viral particles were collected, diluted in PBS, and concentrated by ultracentrifugation in discontinuous 25–50% sucrose gradients. Bands were collected diluted in PBS supplemented with 1% FBS, and virus titers were determined by standard plaque assay on C7/10 cells [44 (link)].
4
Labeling and Tracking Extracellular Vesicles
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ELVs were labelled with Alexa Fluor 555 dye (AF555) conjugated to oligonucleotides (BLOCK-it Alexa Fluor Red Fluorescent Control, Invitrogen, Thermo Fisher Scientific, Vilnius, Lithuania) by lipofection (RNAiMAX, Invitrogen, Thermo Fisher Scientific, Lithuania, Vilnius). Briefly, a mixture of 0.2 µM of AF555-oligonucleotide conjugate was mixed with 3 µL of RNAiMAX reagent in 100 µL of the Opti-MEMTM medium (Gibco™, Thermo Fisher Scientific, Bleiswijk, The Netherlands) and incubated for 5 min at room temperature. Particle preparation (1 mg/mL of total protein) was added into lipofection mixture and incubated at 37 °C for one hour. After incubation, unincorporated dye and residual micelles were removed using Exosome Spin Columns (Invitrogen, Thermo Fisher Scientific, Vilnius, Lithuania). For a micelle-cleaning efficiency assessment, the fluorescence intensity of lipofection mix comprising 0.2 µM pmol AF555-oligonucleotide conjugate, 7.5 uL RNAiMAX reagent and 92.5 uL of PBS was measured before and after cleaning procedure using Tecan Infinite Pro plate reader. The calculated efficiency of unincorporated dye elimination from ELV samples was 99.99% (Figure A2 ). After the labelling and cleaning procedure, the particles were concentrated using 100 K Amicon® ultra centrifugal filters (Merck Millipore, Darmstadt, Germany) and used for internalisation and tracking analysis.
5
Exosome Characterization by Mini-SEC
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To concentrate exosomes isolated by mini-SEC (fraction #4) to 0.5 mL, 100K Amicon Ultra centrifugal filters (EMD Millipore) were used for centrifugation at 4000× g. Vesicles were lysed and were separated on SDS/PAGE gels. Proteins were transferred onto a PDVF Immobilon-P membrane (EMD Millipore) for Western blot analysis. Each lane was loaded with 5 μg of protein from fraction #4, and PVDF membranes were incubated overnight at 4 °C with antibodies specific for TSG101 (1:1000, ab30871, Abcam); Alix (1:1000, ab2171S, Cell Signaling); CD9 (1:500, ab65230, Abcam); CD63 (1:500, ab59479, Abcam); CD81 (1:500, abPA5-13582, ThermoFisher); calnexin (1:2000, ab2433S, Cell Signaling); and Grp94 (1:1000, ab2104, Cell Signaling).
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