The composition of the Chinese botanical formulation KBN, plant parts used, and botanical drug dose are presented in table 1 . The total dosage of KBN is 206 g. Borneol was purchased from Yunnan Linyuan Spice Co., Ltd. (Yunnan, China), the rest of the botanical drugs were purchased from Guangzhou Zhixin Chinese Medicine Pieces Co., Ltd. (Guangzhou, China). Except for borneol, the rest of the botanical drugs were extracted in a 6-and 4-fold volume of 80% ethanol at 100°C for 1.0 h. After filtrating, both filtrates were mixed and concentrated to 1 g/mL raw botanical drugs. Then the concentrated decoction was added 1 g of borneol dissolved in little ethanol to obtain KBN for in vitro experiments. To obtain 0.4, 0.2, and 0.1 g/mL KBN preparation containing 4% Avicel (Dupont, USA) for in vivo experiments, 200, 100, and 50 mL of KBN were respectively added 20 g of Avicel CL-611with stirring (12,000 r/min) and ultrapure water to 500 mL.
Avicel
Manufactured by DuPont
Sourced in United States
Avicel is a microcrystalline cellulose product manufactured by DuPont. It is a fine, white, odorless, and tasteless powder derived from purified, partially depolymerized cellulose. Avicel serves as a binder, disintegrant, and diluent in the formulation of pharmaceutical and dietary supplement products.
Automatically generated - may contain errors
Lab products found in correlation
Rc581 nfdr080i, by DuPont (4 mentions)
Dulbecco modified eagle medium (dmem), by Corning (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Crystal violet, by Merck Group (3 mentions)
Neutral buffered formalin, by Thermo Fisher Scientific (2 mentions)
Fastprep 24 system, by MP Biomedicals (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Buffered formalin, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Optipro medium, by Thermo Fisher Scientific (1 mentions)
Lysing matrix m 2 ml tubes, by MP Biomedicals (1 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
Direct zol rna microprep kit, by Zymo Research (1 mentions)
25 protocols using avicel
1
Composition and Preparation of KBN Botanical Formulation
2
SARS-CoV-2 Viral Titer and Inactivation Assay
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Vero E6 cells were used to determine the titer of our virus stock and to evaluate SARS-CoV-2 inactivation following lysis of infected cells in our HLA-IP buffer. Briefly, we seeded Vero E6 cells into a 12-well plate at a density of 2.5 × 105 cells per well, and the next day, infected them with serial 10-fold dilutions of the virus stock (for titration) or the A549 lysates (for the inactivation assay) for 1h at 37°C. We then added 1 mL per well of the overlay medium containing 2X DMEM (GIBCO: #12800017) supplemented with 4% FBS and mixed at a 1:1 ratio with 1.2% Avicel (DuPont; RC-581) to obtain the final concentrations of 2% and 0.6% for FBS and Avicel, respectively. Three days later, we removed the overlay medium, rinsed the cell monolayer with 1X PBS and fixed the cells with 4% paraformaldehyde for 30 minutes at room temperature. 0.1% crystal violet was used to visualize the plaques.
3
Viral Titer Determination by Plaque Assay
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The titer of our viral stocks was determined by plaque assay. Vero E6 cells were seeded into a 12-well plate at a density of 2.5 × 105 cells per well and infected the next day with serial 10-fold dilutions of the virus stock for 1 h at 37°C. Following virus adsorption, each well was supplemented with 1 mL of overlay media, consisting of 2X DMEM supplemented with 4% FBS and mixed at a 1:1 ratio with 2.4% Avicel (DuPont, Wilmington, DE, USA; RC-581). Three days later, the overlay media was removed, the cell monolayer was washed with 1X PBS and fixed for 1 h at room temperature with 10% neutral buffered formalin (ThermoFisher Scientific). Following formalin removal, fixed cells were then washed with 1X PBS and stained for 1 h at room temperature with 0.1% crystal violet (Sigma-Aldrich) prepared in 10% ethanol/water. After rinsing with tap water, the number of plaques was counted and the virus titer was calculated.
4
SARS-CoV-2 Viral Stock Production
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The isolate BetaCoV/France/IDF00372/2020 (EVAg collection, Ref‐SKU: 014V‐03890) was kindly provided by Sylvie Van der Werf. Viral stocks were produced on Vero‐E6 cells infected at a multiplicity of infection of 1 × 10−4 plaque‐forming units (PFU). The virus was harvested 3 days post‐infection, clarified, and then aliquoted before storage at −80°C. Viral stocks were titrated on Vero‐E6 cells by classical plaque assays using semisolid overlays (Avicel, RC581‐NFDR080I, DuPont) (Baer & Kehn‐Hall, 2014 (link)).
5
Antiviral Screening of Compounds
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A(H1N1)pdm09 (1E6 PFU) and the EC90 (49 µM) concentration of the drugs were incubated for 3 h at 37 °C. The mixture of virus plus molecule was then serially diluted, from 1/9 (5.4 µM) to 1/19000 (2.6 nM), and transferred on MDCK cell, seeded in a 96-well, in serum-free DMEM containing 1% penicillin/streptomycin. After 1 h, the mixture was removed and fresh medium was added. At 16 hpi, viral titers were evaluated by ICC assay and percentages of infection calculated compared to the untreated control, as explained in [20] (link).
SARS-CoV-2 Delta (1E5 PFU) and the maximum non-toxic dose of the molecules (20.7 µM for2 and 31.1 µM for 3 ) were incubated for 3 h at 37 °C. The mixture of virus plus molecule was then serially diluted, from 1/10 (2 µM and 3.1 µM) to 1/1E6 (0.02 nM and 0.03 nM), and transferred on Vero-E6 cells. After 1 h, the mixture was removed and the cells were overlaid with DMEM containing 0.8% Avicel (RC-581-NFDR080I, Dupont). After 72 h of incubation at 37 °C, the cells were fixed with 4% formaldehyde solution and stained with 0.1% crystal violet to count the plaques.
SARS-CoV-2 Delta (1E5 PFU) and the maximum non-toxic dose of the molecules (20.7 µM for
6
SARS-CoV-2 Plaque Assay in VeroE6-ACE2-TMPRSS2 Cells
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Cells were grown in DMEM containing 10% (vol/vol) fetal calf serum (FCS) and incubated at 37°C in 5% CO2. Plaque assays utilized VeroE6 expressing angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) to enhance virus entry [13 (link)]. Serial dilutions of sample were applied to cells for 1 hour at 37°C with rocking. Cells were overlaid with DMEM containing 2% FCS, 1.2% Avicel (DuPont, USA), 50 μg/mL Gentamycin (Fisher Scientific, UK), and 2.5 μg/mL Amphotericin-B (Sigma Aldrich, UK). After 72 hours, the overlay was removed, the monolayer washed, fixed with 100% methanol, stained with 25% (vol/vol) methanol and 0.5% (wt/vol) Crystal Violet, then washed, and plaques enumerated.
7
SARS-CoV-2 NSP1 Quantification and Plasmid Constructs
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Infectious virus particles from cell culture supernatants were quantified by plaque assay. Briefly, Vero-E6 cells were seeded in 12-well cell culture dishes, and once confluent, cells were washed with warm PBS and incubated with dilutions of cell culture supernatants in 100 μL complete DMEM for 1 hr at 37°C/5% CO2. The virus inoculum was then removed, and cells overlaid with 0.6% Avicel (RC-591, Dupont) in DMEM containing 2% HI-FBS. After 48 hr incubation, cells were fixed with 4% paraformaldehyde, and crystal violet (C6158, Merck) staining was done to visualize the plaques.
Plasmids pLVX-EF1alpha-SARS-CoV-2-nsp1-2xStrep-IRES-Puro expressing SARS-CoV-2 NSP1 was a kind gift from Prof. Nevan Krogan (Gordon et al., 2020 (link)). Other plasmids used in this study include Plasmids pRL-TK (mammalian vector for weak constitutive expression of wild-type Renilla luciferase), pGL4 (mammalian vector expressing firefly luciferase), pIFN-β Luc (IFN beta promoter-driven firefly luciferase reporter). The plasmid pMTB242 pcDNA5 FRT-TO-3xFLAG-3C-Nsp1_SARS2 was a kind gift from Prof. Ronald Beckmann.
Plasmids pLVX-EF1alpha-SARS-CoV-2-nsp1-2xStrep-IRES-Puro expressing SARS-CoV-2 NSP1 was a kind gift from Prof. Nevan Krogan (Gordon et al., 2020 (link)). Other plasmids used in this study include Plasmids pRL-TK (mammalian vector for weak constitutive expression of wild-type Renilla luciferase), pGL4 (mammalian vector expressing firefly luciferase), pIFN-β Luc (IFN beta promoter-driven firefly luciferase reporter). The plasmid pMTB242 pcDNA5 FRT-TO-3xFLAG-3C-Nsp1_SARS2 was a kind gift from Prof. Ronald Beckmann.
8
SARS-CoV-2 Virus Stock Preparation and Titration
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SARS-CoV-2 stock was prepared as previously reported.34 (link) Briefly, the SARS-CoV-2 Washington isolate (NCBI accession number: MN985325), obtained from the Centers for Disease Control and Prevention and BEI Resources, was passaged twice on Vero E6 cells to obtain the P2 working stock. For measurement of virus titer, we seeded Vero E6 cells into a 12-well plate at a density of 2.5 × 105 cells per well. The next day, the cells were incubated with serial 10-fold dilutions of the virus stock (200 μl volume per well) for 1h at 37°C, followed by the addition of 1 ml per well of the overlay medium made of 1:1 mixture of 2X DMEM/4% FBS and 1.2% Avicel (DuPont; RC-581). After three days of incubation at 37°C, the overlay medium was removed and the cell monolayer was washed with 1X PBS and fixed with 4% paraformaldehyde. The fixed cells were then stained with 0.1% crystal violet for 1h at room temperature and rinsed with tap water. The numbers of plaques were counted and the virus titer was calculated.
9
SARS-CoV-2 Plaque Assay Protocol
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SARS-CoV-2 infectious clones were propagated on Vero TMPRSS2 cells, sequence verified, and were stored at -80°C until use. For plaque assays, tissue homogenates and cell culture supernatants were analyzed for viral particle formation for in vivo and in vitro experiments, respectively. Briefly, VAT cells were plated and rested for at least 24 hours. Serial dilutions of inoculate of tissue homogenates or cell culture supernatants were added on to the cells. After the 1-hour absorption period, 2.5% Avicel (Dupont, RC-591) was overlaid. After 72 hours, the overlay was removed, the cells were fixed in 10% formalin for one hour, and stained with crystal violet for visualization of plaque formation.
10
SARS-CoV-2 Virus Quantification Assay
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Tissue pieces stored in RNALater were thawed at room temperature and then 22–40 mg of tissue were weighed out and placed into 600 uL of OptiMEM (ThermoFisher Scientific). Tissues were homogenized using a Qiagen TissueLyser II with two dissociation cycles, centrifuged and supernatant was transferred to a new 1.5 mL Eppendorf tube (see “RNA extraction from fLX ” section). Tissue homogenates were then serial diluted (10−1 – 10−6) and 300 uL of supernatant was plated on VeroE6 cells in 12-well plates (2×105 cells/well). Supernatant was incubated for 1 hour at 37°C to allow for viral adsorption then 1 mL of a 1:1 mixture of 2X DMEM 4% FBS 2% penicillin/streptomycin and 2.4% Avicel (Dupont) was overlaid onto each well. Plates were incubated for 72 hours at 37°C with 5% CO2. Avicel was then removed, cells were fixed in 10% buffered formalin (ThermoFisher Scientific) for 1 hour, then stained with 0.1% crystal violet in 10% ethanol/H2O for 30 minutes before washing and quantification.
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