To determine the inhibitory effect of soluble heparin on EV-A71 variants, a virus inactivation assay was performed as previously described [9 (link)]. In brief, viruses were incubated with 2.5 mg/ml of soluble heparin (Sigma, USA) for an hour at 37°C. The treated viruses were inoculated onto pre-seeded RD cells and incubated at 37°C. Two days later, the cell viability of each infected virus variants was measured using CellTiter 96 Aqueous One solution Cell Proliferation Assay (Promega, USA). The relative cell viability was calculated with the following formula:
Soluble heparin
Manufactured by Merck Group
Sourced in United States, Germany
Soluble heparin is a pharmaceutical ingredient used in the production of anticoagulant medications. It is a glycosaminoglycan that functions as an anticoagulant by enhancing the activity of antithrombin, a naturally occurring substance that inhibits blood clotting.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
A488 anti higg, by Jackson ImmunoResearch (1 mentions)
Imagexpress micro xl high content imaging system, by Nikon (1 mentions)
Metaxepress high content image acquisition and analysis software, by PerkinElmer (1 mentions)
Columbus image data storage and analysis system, by PerkinElmer (1 mentions)
Arpe 19 cell, by American Type Culture Collection (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Alexa 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Heparin, by Merck Group (1 mentions)
Heparin sepharose beads, by Merck Group (1 mentions)
6 protocols using soluble heparin
1
Heparin Inhibition of EV-A71 Variants
2
Quantifying VEGF Binding to NRP-1 in ARPE-19 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ARPE-19 cells (ATCC) were incubated for 30 min with serial dilutions of soluble heparin (Sigma-Aldrich; starting from 1000 nM, 1:2) or rhNRP-1-mFc (Regeneron; starting from 100 nM, 1:2) pre-incubated with 10 nM VEGF165 (Regeneron). Bevacizumab was added to the cells (final concentration 10 nM), followed by a 1-h incubation at 37 °C. Surface staining of bevacizumab was detected with A488-anti-hIgG (Jackson Immunoresearch), followed by fixation in 4 % paraformaldehyde and counterstaining with DAPI. Fluorescence was detected by a Molecular Devices ImageXpress Micro XL High-Content Imaging System equipped with a Nikon 20x Plan Fluor ELWD objective lens (NA 0.45; WD 8.2–6.9 mm). All images were acquired with a Molecular Devices 1.4 megapixel cooled CCD camera using MetaXepress® High-Content Image Acquisition and Analysis Software and analyzed using PerkinElmer Columbus Image Data Storage and Analysis System. Figures were made in Adobe Photoshop. No adjustments were made to the original images.
3
Viral Attachment Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Viral attachment to cell surfaces was assessed at 4 °C, which allows for viral binding but precludes entry for many viruses, including enteroviruses39 (link). RD-cell monolayers (2 × 105 cells/well in 12-well plates) were infected with CVA16 (multiplicity of infection [MOI] = 100) in the presence or absence of CHLA (20 µM), PUG (25 µM), soluble heparin (500 µg/ml; Sigma), or a DMSO control (0.25%) for 3 h at 4 °C. The viral inocula were subsequently removed, and the cells were collected into tubes by rinsing with ice-cold flow cytometry buffer (1X PBS plus 2% FBS). After cells were washed twice with ice-cold flow cytometry buffer and fixed with 4% paraformaldehyde for 20 min on ice, the cells were stained with an anti-VP1 antibody (1:2000; Merck-Millipore, Bedford, MA, USA) followed by incubation with a secondary Alexa 488-conjugated anti-mouse IgG (1:250, Invitrogen). Flow cytometry analysis was performed using a BD FACScan flow cytometer (Becton Dickinson; San Jose, CA, USA).
4
Plaque Reduction and Inhibition Assays for FMDV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
(i) Plaque reduction neutralization assay on BHK-21 and WT-CHO cells. The appropriate concentrations of the specific FMDVs (10–50 PFU; plaque forming units) were incubated with the amount (0–1 mg/mL; two-fold serial dilutions) of soluble heparin (Sigma; sodium salt) in phosphate-buffered saline (PBS, pH = 7.4) solution. After 10 min incubation at room temperature (RT) to allow heparin binding to FMDV, the reduction in virus titers of the mixtures was measured by plaque forming assay on BHK-21 cells and WT-CHO cells.
(ii)Plaque inhibition assay on BHK-21 cells. The RGD-containing peptide VR-17 (141-VPNLRGDLQVLAQKVAR-157
[47 (link)]; Invitrogen) was dissolved in PBS containing 1 mM CaCl2 and 0.5 mM MgCl2. Monolayers of BHK-21 cells were pre-incubated with the peptide VR-17 (0–1 mM; ten-fold serial dilutions) for 45 min prior to the addition of viruses for a further 1 h at 37°C. Next, 2 ml of the overlay medium containing 0.6% gum tragacanth were added to the cells. Finally, the cells were fixed with acetone/methanol (1:1) and stained with 0.2% crystal violet at 48 h post-infection. The inhibition of FMDV infection by the RGD-containing peptide VR-17 was calculated by PFU from the infected cell monolayers.
(ii)
[47 (link)]; Invitrogen) was dissolved in PBS containing 1 mM CaCl2 and 0.5 mM MgCl2. Monolayers of BHK-21 cells were pre-incubated with the peptide VR-17 (0–1 mM; ten-fold serial dilutions) for 45 min prior to the addition of viruses for a further 1 h at 37°C. Next, 2 ml of the overlay medium containing 0.6% gum tragacanth were added to the cells. Finally, the cells were fixed with acetone/methanol (1:1) and stained with 0.2% crystal violet at 48 h post-infection. The inhibition of FMDV infection by the RGD-containing peptide VR-17 was calculated by PFU from the infected cell monolayers.
5
Heparin-Binding Protein Pulldown Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Supernatants from Hi-5 adherent cells containing recombinant proteins were incubated with heparin-sepharose beads (Sigma-Aldrich, Germany) for 1.5 h at 4°C. Recombinant proteins were detected with the anti His-tag antibody by Western blotting prior to the pulldown to ensure that similar amounts of all recombinant proteins were used in the assay. The binding of recombinant proteins to the heparin-beads was competed by adding 0.1 to 2 mg of soluble heparin (Sigma-Aldrich, Germany) to the binding reaction. As negative control, agarose beads lacking heparin were used (Sigma-Aldrich, Germany). The beads were washed three times with PBS and the proteins were eluted with denaturalizing loading buffer for SDS-PAGE. The presence of the recombinant proteins was detected by Western blotting using the anti His-tag antibody.
6
Heparin-Induced Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with phosphate-buffered saline (PBS) twice and cultured in serum-free DMEM for 24 h with or without 50 μg/ml soluble heparin (Sigma-Aldrich). The conditioned media were collected, and cell lysates were prepared as described. Loading buffer was added to the conditioned media and cell lysates, which were boiled for 3 min. Then the proteins were separated by SDS–PAGE and analyzed by immunoblot with anti–c-myc and anti–α-tubulin. Quantitation of protein bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!