Cell proliferation following IPA3 treatment was assessed using an MTT assay (Thermo Fisher Scientific, Inc.), as previously described (13 (link)). Briefly, cells were seeded into 48-well cell culture plates, at a density of 5×104 cells/ml per well and incubated at 37°C in a humidified incubator with 5% CO2 for 24 h. Cells were treated with either 10, 20, or 30 µM IPA3 (cat. no. 3622; Tocris BioScience) encapsulated in SSL and SPRL liposomes, empty SSL and SPRL or dimethyl sulfoxide (DMSO) (vehicle) as controls for 24 h. Following this, MTT reagent was added, at a final concentration of 0.25 mg/ml, and the plates were incubated at 37°C for 2 h. After incubation, non-reduced MTT and the medium were aspirated and MTT formazan crystals were dissolved using DMSO. Following an additional 15 min incubation, with constant shaking (2.8×10−3 × g using a Vari-Mix Platform Rocker (Thermo Fisher Scientific, Inc.), plates were read at 590 nm using a Biotek plate reader (Agilent Technologies, Inc.).
Vari mix platform rocker
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Vari Mix Platform Rocker is a laboratory equipment designed for gentle rocking and mixing of samples. It provides a consistent and adjustable rocking motion to facilitate various mixing and incubation applications in the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Biotek plate reader, by Agilent Technologies (1 mentions)
Mtt assay, by Thermo Fisher Scientific (1 mentions)
Rhadamts13, by R&D Systems (1 mentions)
Imaris v9, by Oxford Instruments (1 mentions)
Carboxymethylcellulose, by Merck Group (1 mentions)
Geno grinder, by SPEX (1 mentions)
6 protocols using vari mix platform rocker
1
Cell Proliferation Assay Using IPA3
2
Modulation of ADAMTS13 Activity in FFP
3
Isolation and Propagation of CxFV Virus
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Samples with C t values below 27 (samples 3893: Ct = 26.44; 3897: Ct = 26.77; 3918: Ct = 24.60; 3929: Ct = 26.41) were selected for isolation of CxFV. C6/36 monolayers with 90% confluence were prepared in culture tubes with a 10 cm2 growth area. The initial inoculum of 200 µL of supernatant, filtered through 0.45 µm membranes, was used. After 1 h incubation in rocker platform (Vari Mix Platform Rocker ThermoFisher©) at minimal speed for viral adsorption, the inoculum was removed from the C6/36 cell monolayer, and a fresh L15 culture medium containing 5% FBS was added. Cells were incubated at 28 °C for up to 7 days. Cell culture tubes were evaluated daily for cytopathic effect (CPE). Regardless of the presence of CPE, after seven days of infection, the supernatant was collected, centrifuged for 10 min at 5000 rpm, and 200 µL were used as inoculum for the second passage in C6/36 cells. Isolation was confirmed by qRT-PCR; sample 3929 had the lowest Ct value 16.47 and was chosen to produce a large number of viruses. To achieve a high viral load, sequential passages were performed. Aliquots from both passages were collected for RT-PCR and plaque-forming units (PFU) titration.
4
Visualizing Platelet-Fibrin Clot Formation
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To prepare PRP clots for visualization, 100 μl of PRP was incubated with AF647-conjugated CD42b rabbit anti-human antibody (1 μg/ml; catalog no. 303923, BioLegend, San Diego, CA, USA) to label the platelets, and 1% AF488-conjugated fibrinogen (catalog no. F13191, BioLegend, San Diego, CA, USA) to label fibrinogen for 10 min on a rocker with gentle rocking frequency of approximately 2 Hz (Vari-mix Platform Rocker, Thermo Fisher Scientific, Waltham, MA, USA). After incubation, clots were prepared by adding 20 mM CaCl2 and thrombin (0.2 U/ml). Immediately, 50 μl of the mixture was added on ethanol-cleaned 1-mm microscope slides (Thermo Fisher Scientific, Waltham, MA, USA). PPP clots were prepared in a similar fashion but without the platelet antibody. The clots were formed at 22°C for 45 min. The clots were sandwiched between coverslip and slide and imaged by confocal microscopy with an Apochromat 63× oil objective with a vertical stack interval of 1 μm (Zeiss LSM 700) for quantitative analyses. The images were analyzed using Imaris v9.5 (Oxford Instruments, MA) and National Institutes of Health ImageJ (93 (link)).
5
Viral Titration by Plaque Assay
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For viral titration by plaque-forming units (PFU), monolayers of C6/36 cells were grown in 6-well plates to 80% confluence. The supernatant of the sixth passage of the sample, named 3929 (with higher viral load by qRT-PCR), was serially diluted in base 10 to 106 using L15 medium without FBS supplementation. An inoculum of 700 µL of the dilutions 102 to 106 was used per well. Virus adsorption was carried out for 1 h in a rocker platform (Vari Mix Platform Rocker ThermoFisher©) at minimal speed for viral adsorption. After the adsorption period, 2 mL of a 1:1 mixture of 2% Carboxymethylcellulose (CMC-Sigma) and 2× concentrated L15 medium supplemented with 10% FBS was added to each well. The plate was incubated at 28 °C for 10 days. After incubation, cells were fixed with 10% formaldehyde for at least 24 h and stained with 2% crystal violet. Plaque-forming units were counted for viral titer determination.
6
Liver Sample Extraction and Cleanup
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We adapted a previously reported sample preparation procedure. 24 Chopped and homogenized liver samples (1.0 ± 0.05 g) were transferred to 50-mL polypropylene centrifuge tubes, and an aliquot of 6 mL of extraction solution containing 10% methanol in acetonitrile (v/v) was added to each sample, followed by 10 s vortex and 5 min pulverizing (Geno/Grinder; Spex SamplePrep) at 650 rpm. Samples were then shaken for 30 min (Vari-Mix platform rocker; Thermo Fisher) at a setting of 30, followed by centrifugation at 829 × g for 5 min. The resulting supernatants were further processed using dSPE.
An aliquot of supernatant (5 mL) was transferred to a 15-mL centrifuge tube containing anhydrous magnesium sulfate (175 mg), Florisil PR (100 mg), alumina basic (50 mg), and primary-secondary amine (PSA, 50 mg). The mixture was vortexed for 10 s, then shaken for 30 min followed by centrifugation at 829 × g for 5 min. The cleaned liver extract (4 mL) was carefully transferred to a clean 15-mL centrifuge tube and evaporated to dryness at 45°C under a gentle stream of nitrogen using a nitrogen evaporator (N-Evap; Organomation). The resultant residue was dissolved in 1 mL of methanol by vortexing for 10 s followed by sonication for 5 min prior to filtration through a 0.22-µm (13-mm diameter) PTFE filter (MicroSolv Technology) into a 2-mL amber glass vial for analysis.
An aliquot of supernatant (5 mL) was transferred to a 15-mL centrifuge tube containing anhydrous magnesium sulfate (175 mg), Florisil PR (100 mg), alumina basic (50 mg), and primary-secondary amine (PSA, 50 mg). The mixture was vortexed for 10 s, then shaken for 30 min followed by centrifugation at 829 × g for 5 min. The cleaned liver extract (4 mL) was carefully transferred to a clean 15-mL centrifuge tube and evaporated to dryness at 45°C under a gentle stream of nitrogen using a nitrogen evaporator (N-Evap; Organomation). The resultant residue was dissolved in 1 mL of methanol by vortexing for 10 s followed by sonication for 5 min prior to filtration through a 0.22-µm (13-mm diameter) PTFE filter (MicroSolv Technology) into a 2-mL amber glass vial for analysis.
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