This protocol is designed to verify if a candidate anti HBV compound inhibits viral entry especially by blocking NTCP binding. This technique is intended to investigate if HBV entry or an initial step of infection could be interrupted by any candidate compound. Demonstrating the procedure will be Piyanoot Thongsri, a PSP student from my laboratory.
To begin the measurement of the sodium taurocholate co-transporting polypeptide, or NTCP, incubate the mature hepatocytes with eight mill or more ethylenediaminetetraacetic acid or EDTA for 15 minutes. Collect cell pellets and fix the hepatocytes by adding 300 microliters of 3.7%paraformaldehyde in PBS for 15 minutes. Then transfer the cell suspension into a 1.5 milliliter microcentrifuge tube to centrifuge them at 300 G and 25 degrees Celsius for 10 minutes.
Wash the cells twice using 700 microliters of PBS containing 1%fetal bovine serum or FBS in centrifuge for 10 minutes as described before. Then permeabilize the cells with 300 microliters of 0.05%Triton X 100 in PBS at room temperature for 20 minutes. After the centrifugation give three washes of one minute each using 700 microliters of PBS containing 1%FBS.
Incubate the cells for 30 minutes at four degrees Celsius with the primary antibody against NTCP in a one to 100 ratio, followed by three washes with perm wash buffer, and centrifugation. Stain the cells with a secondary antibody conjugated to Alexa Fluor 488 for 30 minutes at four degrees Celsius and repeat three washes of perm wash buffer for one minute each. After centrifugation at 300 G, resuspend the cell pellet with 200 microliters of PBS containing 1%FBS.
In the software, select the measurement parameters for flow cytometric analysis, including FSC, SSC, FITC, or PE.Set auto compensation adjustment and include the compensation controls as explained in the manuscript. Next, run the unstained sample with a medium fluidic flow rate of 60 microliters per minute. Adjust the forward scatter and side scatter threshold until they cover the cell population of interest.
Mark the gate region in this area and apply to all compensation controls. Set the photo multiplier tube or PMT fluorescence using the unstained control and adjust the PMT voltage of FITC or PE in the negative quadrant. Run the positive stained sample and adjust FITC or PE in the positive quadrant scale.
Run the unstained, FITC stained or PE controls, calculate the compensation, and apply it to the sample settings. Next, run the hepatocyte sample to measure the NTCP level. Next, start analyzing the stained hepatocytes by serially selecting on BD FACSuite windows, the control tube, and then data sources tab.
Wait for the raw data to appear. Next, in the worksheet tab on the right hand side click on the ellipse gate button, select the cell population and SSC and the FSC dot plot using the mouse cursor. Then wait for the circle P1 to appear.
Click on the interval gate button and mark the region in the fluorescein isothiocyanate stained histogram. Starting from the highest fluorescence intensity value to the right hand side. The line P2 appears on the screen.
Click on the experiment tube and observe that the data in the worksheet tab changes. Click on the statistics button and then on the empty space and the worksheet. Then, observe the statistical values of the sodium taurocholate co-transporting polypeptide population.
Aspirate the medium from the 100%confluent MHC well plates and add four micromolar cyclosporine A diluted with William's E Medium for two hours. Next, aspirate the candidate hepatitis B virus entry inhibitor to replace it with one milliliter of Williams E Medium containing 4%polyethylene glycol. Then, incubate the culture medium with hepatitis B virus particles at a multiplicity of infection of 100 per well for 18 hours at 37 degrees Celsius.
Then, discard the supernatant at two milliliters of cold PBS and swirl gently to rinse off the old medium with unbound hepatitis B virus. After culturing the cells with two milliliters of complete William's E Medium for seven days wash the cells with PBS and fix them with 3.7%paraform aldehyde and PBS for 15 minutes at room temperature. Sequentially incubate the infected cells with IF blocking solution for 60 minutes at room temperature in the primary antibody at one to 200 dilution in the blocking solution overnight at four degrees Celsius.
Then give three washes of one minute each using washing solution before incubating the cells with the secondary antibody in one to 500 dilution for one hour at room temperature. After washing thrice, mount the sample with an anti fade mounting medium with DAPI and cover it with a glass cover slip. Using a fluorescence microscope equipped having DAPI and PE filters detect the fluorescence signal with a 20 x objective lens to determine the anti HBV entry activity of the candidate compounds.
For the cell binding assay, prepare the cells as demonstrated before and incubate them with hepatitis B virus particles at four degrees Celsius for two hours. After discarding the medium, rinse the cells with two milliliters of cold PBS with a gentle swirl. Then harvest the infected cells using a cell scraper and disrupt the cells with lysis buffer.
Transfer the cell lysate to a collection tube to extract the total DNA with polymerase chain reaction using the temperature conditions described in the manuscript. After preparing the cells as demonstrated before, incubate the hepatocytes with the sodium taurocholic acid solution for 15 minutes at room temperature, aspirate the solution and wash the cells thrice with cold HEPES. Next, add 300 to 500 microliters of cold HEPES to harvest the cells with cell scrapers on ice.
Homogenize the cells by ultrasonication at 20 kilohertz for 20 seconds, and incubate on ice for five minutes. After centrifugation at 10, 000 G, collect the supernatant and evaluate intracellular taurocholic acid concentration using a taurocholic acid ELISA assay kit. To wash the cells and syringe in the isothermal titration calorimetry or ITC instrument, launch the ITC software.
Under the run experiment highlight tab, select micro cal method for 19 injection dot ITCM and click open. When a new window opens, click on the clean tab and in the cleaning method window select the wash button for cell cleaning method and the rinse button for syringe cleaning method. Click on next and follow the onscreen instructions.
After loading the sample into the ITC, click on the load button present under the run experiment tab. Then click next and follow the video instructions until this loading step is completed. Using a syringe, fill the reference cell with solution buffer in the sample cell with 15 micromolar NTCP in the buffer.
Remove the excess solution, then fill the syringe with a 150 Micromolar Curcumin Ligand solution, avoiding air bubbles. Run the sample by clicking on the run button under the run experiment tab. The experimental information and settings will be displayed on the left hand side.
Enter the ligand protein concentrations and temperature details. Click on start to perform the injection process. Wait for 1.4 hours for the protein ligand interaction to complete.
Once the injection is complete select the highlighted analysis button to analyze the raw data using the analysis software. Then, click on overview to display the raw data and choose the fitting model as one set of binding site. Hepatic maturation features were observed including binucleated cells and polygonal shaped morphology, especially in the differentiated stage of MHC.
A large increase in NTCP expression was observed in differentiated HepaRG and differentiated MHC. The highly glycosylated form of NTCP was detected more in differentiated MHC than in differentiated HepaRG. The NTCP levels were higher in differentiated MHC cells than in the undifferentiated cells.
Virus immunofluorescence staining evaluated the anti HBV entry activities on day seven post-infection and the level of virus binding on the cell surface receptor was evaluated by real-time polymerase chain reaction. Taurocholic acid uptake analysis suggested that the punitive inhibitory activity of candidate compounds decreased Hepatitis B virus binding through NTCP. The colorimetric alteration was detected with continuous injections to the sample cell.
A standard non-linear least squares regression was plotted based on one binding site fitted well to the data. The solid line indicated the best fit to the experimental values For SBB binding as a and that cells and hepatitis B viral particle are incubated at four degrees Celsius. Taurocholic acid update can be evaluated using the radioactive technique.
The level of radioactive taurocholic acid can be quantified using a liquid simulation counter. This technique is simple and quick to screen for viral entry in inquiry activity of any antiviral regimen that would help prevent either infection or reinfection.
