At each infection time point, 150 µL of 1x dPBS was added and incubated in the apical chamber for 10 min at 35 °C to recover progeny viruses as the apical wash. The plaque assay for viral quantification was performed using overnight MDCK cultures (ATCC, Manassas, VA, USA) at 85–95% confluence in 24-well plates. The MDCK cells were incubated with 100 µL of serial dilutions (from 10−1 to 10−6) of virus from apical washes at 35 °C for 1 h, where plates were rocked every 15 min to ensure equal viral distribution. After incubation, the inocula were removed and replaced with 1 mL of Avicel (FMC Biopolymer, Philadelphia, PA, USA) overlay, and incubated at 35 °C for 65–72 h. After incubation, Avicel overlay were removed, and cells were fixed with 4% formaldehyde in 1× PBS for 1 h. Formaldehyde was then removed, and cells were washed with 1× PBS prior to staining with 1% crystal violet for 15 min before washing the stain away. The plaque-forming units (PFU) were calculated as follows: Number of plaques × dilution factor = number of PFU per 100 µL.
Avicel
Manufactured by FMC Biopolymer
Sourced in United States, Belgium
Avicel is a microcrystalline cellulose product manufactured by FMC Biopolymer. It is a naturally derived, purified, and highly crystalline form of cellulose. Avicel serves as a functional excipient in the pharmaceutical and food industries, providing various properties such as binding, disintegrating, and texturizing.
Automatically generated - may contain errors
Lab products found in correlation
Minimum essential medium (mem), by Thermo Fisher Scientific (5 mentions)
Crystal violet, by Merck Group (4 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (4 mentions)
Nacho3, by Thermo Fisher Scientific (3 mentions)
Hepes solution, by Thermo Fisher Scientific (3 mentions)
Toluidine blue, by Merck Group (3 mentions)
Trueblue peroxidase substrate, by LGC (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Bead mill, by Analytik Jena (2 mentions)
Tpck treated trypsin, by Worthington (2 mentions)
Nahco3, by Thermo Fisher Scientific (2 mentions)
Crystal violet, by Boston BioProducts (2 mentions)
Paraformaldehyde (pfa), by Boston BioProducts (2 mentions)
Pen strep amp b solution, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Tpck treated trypsin, by Merck Group (2 mentions)
Crystal violet solution, by Merck Group (2 mentions)
Neutral buffered formalin, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Whirl pak sampling bags, by Merck Group (2 mentions)
104 protocols using avicel
1
Quantification of Progeny Viruses
2
Quantifying Viral Titers: HRV1A and H1N1pdm Assays
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The HRV1A titers were determined in a 24-well cell culture plate containing subconfluence (90–95%) HeLa cell monolayers using plaque assay. Briefly, the HRV1A virus inoculum was diluted 1 : 10 in PBS++/BA. The growth media from HeLa cells were removed and the cell monolayers were washed using PBS++. The diluted virus was added to each well and incubated for 1 h at 33°C and 5% CO2 and then removed. 500 μl of Avicel-media containing 1x MEM and 1.25% Avicel (FMC BioPolymer, Belgium) was added and cells were further incubated for 48 h at 33°C with 5% CO2. After removal of the overlay media, the cell monolayers were washed with PBS++, stained with 0.1% crystal violet solution, further washed, and dried. The plaques corresponding to propagating viral particles were counted. The H1N1pdm titers were determined in MDCK-II using focus assay. To analyze the binase effect against H1N1pdm or HRV1A and identify the EC50 of binase, the plaques were counted and PFU/ml was measured using MS Excel Software package. The PFU/ml values of the binase nontreated infected HeLa cells were used as 100%. The EC50 was determined using GraphPad Prism 5.0 Software (GraphPad Software Inc., USA) by plotting the percentage of viable cells as a function of the compound concentration.
3
Quantification of Viral Titers from hNEC Co-Cultures
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The apical RPMI medium was collected upon 24 and 48 h after co-culture establishment for viral quantification. MDCK cells at 85–95% confluence in 24-well plates were incubated with 100 μL of serial dilutions from 10−1-10−4 of virus from infected hNECs at 35°C for 1 h. The plates were rocked every 15 min to ensure equal distribution of virus. The inoculums were removed and replaced with 1 mL of Avicel (FMC Biopolymer) overlay on each well and incubated at 35°C with 5% CO2 for 65–72 h. Avicel overlay was removed after the incubation period and cells were fixed with 4% formaldehyde solution in 1 × PBS for 1 h. Formaldehyde solution was removed and cells were washed with 1 × PBS. The fixed cells were stained with 1% crystal violet for 15 min and washed with running water. The plaque-forming units (PFU) were calculated as follows: Number of plaques × dilution factor = number of PFU per 100 μL.
4
Screening RVFV Clone 13 Inhibitors
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Vero B4 cells (120,000 per well) were seeded in 12-well plates 1 day before infection. Cells were infected in triplicate with RVFV clone 13 (40 PFU/well) 19 and incubated for 1.5 h at 37 °C. Virus was removed, 600 µL of Avicel overlay (300 µL of 2 × cell culture medium + 300 µL of 2.4% Avicel [FMC BioPolymer, Philadelphia, PA]) containing hit compounds (12.5 and 3.12 µM) was added, and the plate was incubated at 37 °C for 72 h. Then, the overlay was removed and the wells were washed with PBS. To count the plaques, cells were fixed with 4% paraformaldehyde for 1 h at 20 °C and then stained with 1% crystal violet.
5
SARS-CoV-2 VOC Plaque Assay
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Samples were serially diluted (tenfold dilutions starting at a 1:10 initial dilution) in DMEM medium supplemented with 2% FBS containing Antibiotic-Antimycotic (Gibco). The serial dilutions were added to Vero E6 cells seeded in 12-well plates at 3 × 105 cells per well 24 hours before. The virus was allowed to adsorb for 1 hour at 37 °C. Subsequently, the inoculum was removed, and the cells were overlaid with 1.2% Avicel (FMC BioPolymer) in DMEM and incubated for 3 days at 37 °C with 5% CO2. After three days, the Avicel was removed, cells were washed once with PBS, fixed with 10% neutral buffered formalin, and plaques were visualized using 1% crystal violet. For dwarf hamsters infected with VOC gamma, delta, and omicron, plaque assays were performed with VeroE6-TMPRSS2 cells.
6
Inhibition of Bluetongue Virus Infection
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HeLa cells were grown in 12-well plates. Cells were either pretreated for 2 or 1 h postinfection with 300 nM geldanamycin or 1 or 10 μM VER-155008 or DMSO control. Cells were infected with BTV1 or the supernatant of previously harvested cells. After adsorption for 30 min at 4°C, the cells were incubated at 37°C in growth medium for 1 h in the presence of inhibitor. The growth medium was removed and replaced with 0.6% Avicel (FMC BioPolymer) overlay medium (Eagle minimal essential medium containing l -glutamine, 10% FBS, and antibiotics) in conjunction with inhibitors, where appropriate. Cells were incubated at 37°C for 72 h before being fixed with 4% paraformaldehyde and subsequently stained with crystal violet. Titers are expressed as PFU/ml.
7
EV-D68 Virus Plaque Assay Protocol
8
Plaque Assay for SARS-CoV-2 Quantification
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Vero E6 cells were infected with serial dilutions of virus and incubated under an overlay consisting of DMEM supplemented with 2% FCS and 0.6% Avicel (FMC BioPolymer) at 37°C for 5 days. Cell monolayers were fixed with 4% formaldehyde. Following fixation, cell monolayers were stained with Giemsa to visualize plaques.
9
Plaque Assay for SARS-CoV-2 in VeroE6 Cells
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VeroE6 cells were seeded in six-well clusters at a density of 3.5 × 105 cells/well in 2 ml/well of DMEM–8% FCS, followed by overnight incubation at 37°C. Samples were 10-fold serially diluted in EMEM–2% FCS, and 500 μl/well of each dilution was used to infect confluent monolayers of VeroE6 cells for 1 h at 37°C on a rocker. The inoculum was removed and replaced with 2 ml/well of an overlay containing DMEM, 1.2% Avicel (FMC BioPolymer), 2% FCS, 50 mM HEPES, and antibiotics. After a 3-day incubation, monolayers were fixed with 3.7% formaldehyde in PBS, and plaques were visualized using crystal violet staining.
10
Virus Particle Activation and Plaque Assay Protocol
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Virus samples were diluted with 1 ml infection medium (DMEM with 0.1% FCS, 0.2% BSA, dissolved in H2O), penicillin [100 U/ml]/streptomycin [100 μg/ml]. To activate virus particles with uncleaved HA, infection medium in some experiments also contained 2 μg/ml TPCK-trypsin.
Plaque assays were performed on MDCK II cells in six well plates. Cells were infected with serial 10-fold dilutions of virus, incubated for 1 h at 37 °C, washed with PBS and overlaid with 1.25% Avicel (FMC BioPolymer), 1% NaHCO3, 0.1% FCS, 0.2% BSA (dissolved in H2O) and 2 μg/ml TPCK-trypsin. After incubation for 48 h at 37 °C the cell cultures were fixed with 4% PFA, cells were stained with 0.1% crystal violet, and the plaques were counted.
In the case where trypsin was not present in the infection medium, it was also excluded from the overlay medium. Instead, 5 h post infection, a second layer of overlay medium containing 2 μg/ml TPCK-trypsin was added to activate progeny virus and allow plaque formation.
HA assays were performed in 96 well U-bottom microwell plates using 50 μl PBS, 50 μl serially diluted virus samples and 50 μl 1% chicken red blood cells. The results were recorded after 30 min of incubation at room temperature.
Plaque assays were performed on MDCK II cells in six well plates. Cells were infected with serial 10-fold dilutions of virus, incubated for 1 h at 37 °C, washed with PBS and overlaid with 1.25% Avicel (FMC BioPolymer), 1% NaHCO3, 0.1% FCS, 0.2% BSA (dissolved in H2O) and 2 μg/ml TPCK-trypsin. After incubation for 48 h at 37 °C the cell cultures were fixed with 4% PFA, cells were stained with 0.1% crystal violet, and the plaques were counted.
In the case where trypsin was not present in the infection medium, it was also excluded from the overlay medium. Instead, 5 h post infection, a second layer of overlay medium containing 2 μg/ml TPCK-trypsin was added to activate progeny virus and allow plaque formation.
HA assays were performed in 96 well U-bottom microwell plates using 50 μl PBS, 50 μl serially diluted virus samples and 50 μl 1% chicken red blood cells. The results were recorded after 30 min of incubation at room temperature.
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