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Experiments

Plasmid Construction

plasmid-design

We conducted the design of plasmids involved in this project exclusively using Benchling, a platform for molecular biology research and design.

procurement of DNA

For this project, we acquired synthetic DNA as follows: All oligoDNA was purchased from Eurofins Scientific. Other synthetic gene fragments were sourced from gBlocks by Integrated Gene Technologies and Gene Fragments by TWIST BIOSCIENCE.

PCR

The following procedure was used to amplify DNA.
  1. The sample was prepared as follows:
      2. Component Amount(μL)
      Template (diluted to 0.1~1 ng/μL) 1
      Primers (10 uM each) 0.4
      2x KOD One Blue 10
      Milli-Q water 8.6
      Total 20
    KOD ONE Blue was provided by Toyobo.
  2. PCR was performed with the following thermal cycling conditions:
      3. Step Temperature (°C) Time(s) Repetitions
      Denaturation 98 10 29
      Annealing 55 5 29
      Extention 68 ≤1 kb: 1
      1~10 kb: 5 / kb      		 29
      Hold 4 pause --

Gel Preparation

Gels were created for electrophoresis as follows:
  1. Agarose was dissolved in 1x TAE buffer at a rate of 1% w/v and the mixture was heated until agarose dissolved.
  2. The mixture was poured into a tray with a comb.
  3. EtBr was added at a 1:10 ratio to the gel, and gently mixed to ensure even distribution.
  4. The gel was allowed to solidify at room temperature.
1x TAE buffer had 40 mM tris-acetate, and 1 mM EDTA. 10 mg/mL EtBr was purchased from Takara Bio Inc.

Gel Electrophoresis

This procedure was conducted to assess the length of DNA fragments which is amplified through techniques such as PCR.
  1. The gel was placed in the electrophoresis chamber without removing it from the tray.
  2. 1x TAE buffer was poured into the chamber until the gel was fully immersed.
  3. An appropriate amount of size marker was added to the first lane.
  4. The samples (3 μL each) were sequentially loaded in the lanes, starting from the second lane.
  5. The chamber was turned on and electrophoresis was run at 100 V for 20 minutes.
  6. After, the gel was carefully removed from the tray and transferred to another large, transparent tray.
  7. The bands were visualized using a CCD camera.
Two kinds of size markers were used: λ styI, λ phage DNA fragments cleaved with restriction enzyme EcoT14I, and a mixture of TOYOBO's 100bp ladder and 6x loading dye. 10 μL of the former and 6 μL of the latter were used. The CCD camera was part of the ChemiDoc XRS+ System purchased from Bio-Rad Laboratories.

Dpnl Treatment

DpnI treatment was performed to cleave plasmids propagated in E. coli when they were used as PCR templates. This prevents any residual E. coli-derived plasmid from being carried over.
  1. 1 μL of DpnI was added to 20 μL of the PCR product.
  2. The mixture was incubated at 37°C for a minimum of 1 hour.
DpnI was purchased from Takara Bio Inc.

DNA Purification

DNA purification was conducted to eliminate excess enzymes and residual substances after PCR.
  1. 20 μl of the PCR product was mixed with 100 μl Buffer PB in a 1.5 mL tube.
  2. This mixture was transferred to a silica membrane column and centrifuged at 14,000 rpm for 1 minute.
  3. The supernatant was discarded.
  4. 700 μl of Buffer PE was added to the column and centrifuged at 14,000 rpm for 1 minute at room temperature.
  5. The supernatant was discarded.
  6. The upper portion of the column was attached to a new 1.5 mL tube.
  7. DNA was eluted with 10 μl of Milli-Q water.
  8. The mixture was left for 1 minute and then centrifuged at 14,000 rpm for 1 minute.
  9. Purified DNA was obtained in the 1.5 mL tube.
Silica membrane columns were purchased from Aji Bio-Pharma Oligos. Buffer PB and PE were purchased from QIAGEN.

DNA Concentration Measurement

To measure the concentration and purity of purified DNA, we used the NanoDrop One spectrophotometer from Thermo Fisher Scientific.
  1. The instrument was turned on, and the arm was raised.
  2. The platform was wiped with a Kimwipe which is wet with Milli-Q water, and dried by dabbing with a dry Kimwipe.
  3. A blank measurement was taken using 1 μL of the same buffer used for the DNA solution.
  4. The concentration and purity of the DNA were measured with 1 μL of the DNA solution.
  5. After every measurement, the platform was again wiped with a Kimwipe wet with Milli-Q water and dried by dabbing.
  6. After all measurements were taken, the arm was lowered, and the instrument was turned off.

DNA Assembly

We utilized the XE cocktail method (Liu et al., 2023), a low-cost PCR enzyme developed by former members of our team, to combine DNA fragments [1].
  1. The thermal cycler was preheated to 37°C.
  2. The samples were prepared as follows:
      3. Component μL
      Inserts (diluted to 10~30 ng/μL) 1 each
      Vector 1
      2x XE cocktail total volume of other components
  3. The cocktail was immediately transferred to the thermal cycler and the following program was run.
      4. Step Temperature (°C) Time(min)
      1 37 pause
      2 37 5
      3 65 5
      4 4 pause
  4. [1]Liu, A.Y., Koga, H., Goya, C., Kitabatake, M. (2023). Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors. Genes to Cells, 28(8), 553-562. https://doi.org/10.1111/gtc.13034

Transformation into E.coil

We introduced the plasmid into E. coli following this protocol.
  1. Up to 2 μl of the assembly product or plasmid was added to 10 μl of competent cells.
  2. The mixture was left on ice for 20 minutes.
  3. The mixture was placed in a heat block at 42°C for 45 seconds.
  4. The mixture was left on ice for 1 minute.
  5. 100 μl of SOC medium was added.
  6. The mixture was incubated at 37°C for 1 hour in the case of an assembly product, and 45 minutes for the plasmid.
  7. The liquid was spread onto a plate following aseptic techniques.
  8. The plates were labeled and incubated at 37°C overnight.

Three strains of competent cells were used in this experiment: DH5α, BL21, and BW25113. DH5α was purchased from TOYOBO, and BL21 (DE3) from COSMO BIO CO., LTD. The BW25113 strains were prepared in-house.

Direct Colony PCR

We performed Direct Colony PCR to confirm the presence of the desired plasmid within the colonies grown on an agar plate.
  1. The samples were prepared as follows:
      2. Component Amount(μL)
      Primers (diluted to 10 mM) 0.3
      2X KOD ONE Blue 5
      Milli-Q water To 10
      Total 10
  2. A single, relatively isolated colony was picked using a pipette tip, and the tip was dipped into the liquid which is prepared as the above in a PCR tube.
  3. The DNA was amplified using the following thermal cycling conditions:
      4. Step Temperature (°C) Time(s) Repetitions
      Pre-heating 98 30 1
      Denaturation 98 10 29
      Annealing 55 5 29
      Extension 68 ≤1 kb: 1
      1~10 kb: 5 / kb      		 29
      Hold 4 pause --

Inocuration

To cultivate a significant quantity of E. coli that is believed to have incorporated the correct plasmid from direct colony PCR, we inoculated them in test tubes with LB medium.
  1. 3 mL of LB medium with the appropriate antibiotic was prepared in a test tube.
  2. A single colony was picked using a pipette tip and suspended in the prepared medium.
  3. The test tube was incubated at 37°C and agitated 180 rpm for at least 16 hours in a shaker.

Miniprep

With this method, we extracted the desired plasmid from E. coli cells and purified it. For this procedure, we used the Promega Wizard Plus SV Minipreps DNA purification System.
  1. 1.5 mL of the 3 mL culture from the test tube was decanted into a 1.5 mL tube.
  2. The 1.5 mL tube was centrifuged at 13,000 rpm for 1 minute, and the supernatant was discarded.
  3. Steps 1 and 2 were repeated.
  4. The 1.5 mL tube was centrifuged at 13,000 rpm for 1 minute.
  5. The supernatant was removed with a pipette.
  6. The precipitated cells were fully resuspended with 250 μL of CRA, vortexing, and pipetting.
  7. 250 μL of CLA was added, and the solution was mixed with gentle inversion.
  8. Once the solution became clear, 10 μL of Alkaline protease was added, and the solution was mixed with inversion. The solution was left for 5 minutes.
  9. 350 μL of NSB was added and mixed well by vortexing.
  10. The mixture was centrifuged at 13,000 rpm for 10 minutes.
  11. An SV minicolumn was placed over a collection tube, and the supernatant was transferred into it.
  12. The minicolumn and tube were centrifuged at 13,000 rpm for 1 minute.
  13. The supernatant in the collection tube was discarded, and 750 μL of Column Wash Water was added to the tube.
  14. The mixture was centrifuged at 13,000 rpm for 10 minutes.
  15. The supernatant in the collection tube was discarded, and 250 μL of Column Wash Water was added to the tube.
  16. The mixture was centrifuged at 13,000 rpm for 1 minute.
  17. The supernatant in the collection tube was discarded, and the collection tube and column were again centrifuged at 13,000 rpm for 2 minutes.
  18. The SV minicolumn was placed on the top of a 1.5 mL tube, and DNA was eluted with 50 μL of Milli-Q water.
  19. The mixture was centrifuged at 13,000 rpm for 1 minute.
  20. The DNA was obtained from the 1.5 mL tube.

Sequencing

We conducted sequence analysis using Sanger sequencing provided by Azenta Life Science. All functional regions of the plasmid used in this study were subjected to sequence analysis to verify their sequences.

Medium Composition

LB liquid medium

Luria broth medium was used as the medium for the culture of E. coli.
  1. The following compounds were mixed in a large glass jar.
      2. Component Amount
      Tryptone 10g
      Yeast Extract 5g
      NaCl 5g
      RO Water 1L
  2. This mixture was autoclaved at 121°C for 15 minutes to sterilize.
  3. When it cooled to around 60°C, the appropriate antibiotics were added.

LB Agar Plate Preparation

We prepared LB agar medium to create plates for culturing E. coli.
  1. A mixture was prepared in a large glass jar as follows:
      2. Component Amount
      Tryptone 10g
      Yeast Extract 5g
      NaCl 5g
      Agar 12g
      RO Water 1L
  2. The mixture was autoclaved at 121°C for 15 minutes to sterilize.
  3. When the mixture cooled to around 60°C, it was mixed thoroughly and the appropriate antibiotics were added to it.
  4. ~20 mL of the agar medium was poured into each plate and allowed to solidify.

Pull-Down Assay

Pull-Down Assay

  1. Add 1 ml of Ni-NTA beads to a 50 ml tube and wash 3 times with Wash Buffer.
  2. Adjust the washed beads to 5 ml, then aliquot 250 μl into 1.5 ml tubes.
  3. Centrifuge at 13,000 rpm for 1 minute and remove the supernatant.
  4. Add the Target DNA and Standard DNA.
  5. Add His-tagged protein and adjust to 1 ml with Reaction Buffer.
  6. Take 20 μl of the input sample and store it.
  7. Incubate at 4°C for 30 minutes.
  8. Centrifuge at 13,000 rpm for 1 minute.
  9. Remove the supernatant and wash the beads 3 times.
  10. Add 100 μl of Milli-Q water to both the input sample and beads, then boil at 95°C for 5 minutes.
  11. Centrifuge at 13,000 rpm for 10 minutes.
  12. Store the supernatant.

Ligation

Ligation

Component volume(μL)
Ligation high 2.5
Vector 1
Insert 1.5
total 5

SDS-PAGE

SDS-PAGE

Component volume(ml)
40%Acrylamide 3.125
1M Tris pH 8.8 3.75
10% SDS 0.1
DW 3.025
total 10
Add 100 μL of 10% APS and 10 μL of TEMED, and mix well.
Preparation of the Stacking Gel
Component volume(ml)
40%Acrylamide 1
1M Tris pH 8.8 1.25
10% SDS 0.1
DW 7.65
total 10
Add 100 μL of 10% APS and 10 μL of TEMED, and mix well.

Cell-related experiments

Thawing frozen cells

Thaw cells that have been frozen at -80°C. Work under disinfect conditions to prevent cell contamination.
  1. Add 5 mL of Dulbecco's Modified Eagle Medium (DMEM(-/-)) to the tube and warm it at 37°C.
  2. Remove the cells from -80°C, thaw them at 37°C, and then sterilize them with ethanol.
  3. Suspend the cells in tube 1 and centrifuge at 800 rpm for 5 minutes at room temperature.
  4. Wash once with DMEM (-/-)
  5. The supernatant was aspirated and warmed to 37°C. Add 100 μl of complete DMEM medium (hereinafter referred to as DMEM) supplemented with 10% FBS, 1% P/S, and 1% L-Glutamine and suspended.
Composition of complete DMEM supplemented with 10% FBS, 1% P/S, 1% L-Glutamine
Reagent Volume [ml]
DMEM(-/-) 352
Fetal Bovine Serum (FBS) 40
Penicillin/Streptomycin (P/S) 4
L-Glutamine 4
  1. Seed the cells on a new plate, gently mix to evenly distribute them, and place in an incubator (HEK293: 37℃, DF-1: 38.5℃).

Cell passaging

To maintain healthy cells and promote their proliferation, subculture the cells at the appropriate time when they reach confluence. Work under sterile conditions to prevent cell contamination.
  1. Warm DMEM and Phosphate Buffered Saline (PBS) at 37°C and warm Trypsin/EDTA at room temperature.
  2. Remove the cells from the incubator and aspirate the old medium from the plate or flask.
  3. Wash with PBS
  4. Add trypsin/EDTA and incubate until cells detach (HEK293: 37℃, DF-1: 38.5℃).
  5. Add DMEM to neutralize the trypsin and collect the cells in a tube.
  6. (Optional) Take a small amount of the cell suspension from step 5 and count the number of cells.
  7. Centrifuge HEK293 at 900 rpm for 5 minutes, and DF-1 at 1200 rpm for 5 minutes.
  8. Aspirate the supernatant and add DMEM to suspend the cells.
  9. Add the appropriate amount of DMEM and cells to a new plate or flask, mix gently to evenly distribute, and place in an incubator (HEK293: 37°C, DF-1: 38.5°C).

Transfection Using Lipofectamine 2000 Reagent

  1. Seed cells into appropriate well plates 24 hours prior to transfection and incubate at 37°C for HEK293 and 38.5°C for DF-1 (cells should reach 70-90% confluence).
  2. Dilute Lipofectamine 2000 Reagent in Opti-MEM Medium
  3. Dilute DNA in Opti-MEM Medium in a separate tube from step 2.
  4. Mix the solutions from step 2 and step 3 in a 1:1 ratio and incubate at room temperature for 5 minutes.
  5. Add the DNA-lipid complexes to the cells and return the cells to the incubator.
Component 96-well 24-well 6-well
DNA-lipid complex per well 10 μL 50 μL 250 μL
Final DNA used per well 100 ng 500 ng 2500 ng
Final Lipofectamine® 2000 Reagent used per well 0.2-0.5 μL 1.0-2.5 μL 5.0-12.5 μL

Transfection Using FuGENE 6 Transfection Reagent

  1. Seed cells into appropriate well plates 24 hours prior to transfection and incubate at 37°C for HEK293 and 38.5°C for DF-1 (cells should reach 50-80% confluence).
  2. Add Fugene 6 Reagent to Opti-MEM Medium and incubate at room temperature for 5 minutes.
  3. Add plasmid DNA and incubate for 15 minutes.
  4. Add the DNA-FuGENE complex to the cells and return the cells to the incubator.
Component 96-well 24-well 6-well
DNA-FuGENE complex per well 5 μL 25 μL 100 μL
Final DNA used per well 100 ng 500 ng 2000 ng
Final Lipofectamine® 2000 Reagent used per well 0.2-0.5 μL 1.0-2.5 μL 5.0-12.5 μL

Creation of Stable Cell Lines

Cell lines that stably express the target protein are generated.
  1. Transfecting cells with DNA
  2. Incubate for 1-2 days
  3. Remove the medium from the plate and add DMEM containing 400 μg/mL of G418 to the plate containing the cells.
  4. Maintain the cells in DMEM containing 400 µg/mL of G418 at least 2 weeks.

Cellular protein harvest

Protein recovery from cells for SDS-PAGE and Western blotting. Protocol for 96-well plates is shown.
  1. Wash the cells with 50 ul/well of ice-cold PBS(-).
  2. Add 20 ul/well of ice-cold sample buffer, and transfer all the cells to a PCR tube while scraping the bottom of the dish with a pipette.
    2X sample buffer:
    125mM Tris-HCl buffer(pH6.8)
    4% SDS
    40% Glycerol
    0.01% BP
    10% b-mercaptoethanol (add immediately before use)
  3. Wash the well with 10 ul of sample buffer, add to the tube from step 2, vortex, and spin down.
  4. Place the spun down sample in a thermal cycler and treat at 95°C for 5 minutes.
  5. Store protein samples at -80 °C

Western blot

Gel making
  1. Place the gel plate in the gel preparation bag, clamp it on both sides with acrylic plates, secure the spacer with a clip to prevent it from coming off, and then mark with a pen the area that will be 1 cm below the bottom of the comb when it is inserted.
  2. Create a running gel. Mix Acrylamide, Tris, and Milli-Q and vortex. Then add APS and TEMED and mix again.
Component Volume (10%) Volume (12.5%) Volume (15%)
30% AA 3.3 mL 4.2 mL 5.0 mL
1M Tris (pH8.8) 3.75 mL 3.75 mL 3.75 mL
10% SDS 0.1 mL 0.1 mL 0.1 mL
DW 2.817 μL 2.817 μL 2.817 μL 2.817 μL
Total About 10.0 mL About 10.0 mL About 10.0 mL About 10.0 mL
  1. Slowly pour the gel through the gap in the gel preparation bag until the liquid level reaches the pen mark, then slowly add 100 ul Water-saturated butanol from above and let it stand for 20 minutes.
  2. Aspirate Water-saturated butanol
  3. Prepare the stacking gel in a 15 ml tube with the following composition, similar to the running gel:
Component Volume
30% Acrylamide 0.4 mL
0.5M Tris (pH6.8) 0.75 mL
Milli-Q 1.85 mL
10% APS 20 μL
TEMED 2 μL
Total About 3.0 mL
  1. Place the stacking gel on top of the running gel (approximately 2.4 mL), insert the comb, and let it stand for 30 minutes.
  2. Remove the gel from the gel preparation bag while pouring distilled water over it, taking care not to trap bubbles in the gel, and rinse it lightly with distilled water.
Electrophoresis
  1. Add 2X sample buffer and 1X PBS in a 1:1 ratio and mix (1X sample buffer).
  2. Thaw the protein sample on ice and mix the protein with the sample buffer.
  3. Remove the comb from the gel plate, remove any gel adhering to the sides, and place the gel plate in the holder with the notched side facing inward.
  4. After placing the gel plate in the container, pour 1X running buffer onto the outside of the gel plate by about 5 cm, and tilt the container to remove any bubbles at the bottom of the gel cassette.
  5. Pour 1X running buffer into the gel plate, filling it up to the mark on the outside of the container.
  6. Apply sample and ladder to wells
    To prevent smiling, add an equal amount of 1X sample buffer to each empty well.
  7. Run at CV (constant voltage) 300V for 15 minutes.
Blotting
  1. Pour methanol into a Tupperware container and soak the PVSF membrane cut into 45 x 88 mm pieces for about 20 seconds.
  2. Transfer the membrane to 1X transfer buffer and shake for at least 15 minutes.
  3. Place two pieces of filter paper cut to 45 x 88 mm between the sponges, soak them in 1X transfer buffer in a large Tupperware container, and leave them to stand for at least 5 minutes.
  4. After electrophoresis is complete, disassemble the gel plate, cut off the stacking gel and the bottom of the gel, place them in a Tupperware container containing 1X transfer buffer, and shake for 5 minutes.
  5. On a large tray, place the layers of material onto the cathode core of the transfer device in the following order: sponge, filter paper, gel, membrane, filter paper, sponge, taking care not to trap air bubbles from below, and finally place the anode core of the transfer device on top.
  6. Place the transfer device horizontally into the electrophoresis chamber, fix it with a wedge, then turn it vertically and fill it up with a new 1X transfer buffer.
  7. Chill on ice and run at CV (constant voltage) 30V for 15 minutes.
Blocking and primary antibody
  1. Prepare a blocking solution (5% BSA) by dissolving 0.5 g of BSA (for blocking) in 10 mL of PBST.
  2. Add 2 mL of blocking solution to 8 mL of PBST to make antibody dilution solution (1% BSA).
  3. After the transfer is complete, pour 10 mL of PBST into a Tupperware container, immerse the membrane with the transferred side facing up using tweezers, and shake on a shaker at room temperature for 10 minutes.
  4. Pack the membrane in 3 mL of blocking solution using plastic film or a 25 mL tube and rotate at room temperature for 1 hour.
  5. Dilute the primary antibody to an appropriate concentration in an appropriate amount of antibody diluent (minimum 3 mL for a 25 mL tube), pack the membrane removed from blocking in the same manner as blocking, and rotate it at 4°C overnight.
Secondary antibody and ECL reaction
  1. Pour 10 mL of PBST into a Tupperware container, immerse the membrane in it, and shake it at room temperature for 10 minutes. Repeat this process three times.
  2. Dilute the secondary antibody in antibody diluent to an appropriate concentration (Cytiva antibodies are 1:10,000), pack the membrane in the same manner as for blocking, and rotate at room temperature for 1 hour.
  3. Pour 10 mL of PBST into a Tupperware container, immerse the membrane in it, and shake it at room temperature for 10 minutes. Repeat this process three times.
  4. Mix ECL reagent A and B in a 1:1 ratio in an Eppendorf tube.
    *Required ECL mixture is 50–100 µl/cm²
  5. Place the membrane in a plastic container for measuring, add the ECL mixture and shake gently to spread it evenly.
  6. Place the membrane in the scanner with the protein-adsorbed side facing down, absorb excess ECL mixture, and then scan.

Virus Infection Experiments

Collagen Coating

  1. Prepare collagen solution and add 50 µL/well to 96-well plates.
  2. Incubate at room temperature on a shaker for at least 1 hour.

Cell Preparation 1

Use DF-1 cells:
  1. Aspirate 10% FBS/DMEM from flasks.
  2. Wash twice with PBS.
  3. Add trypsin and incubate at 38.5°C for 5 min.
  4. Add 2 mL DMEM and resuspend cells by pipetting; transfer to 5 mL tubes.
  5. Centrifuge at 1200 rpm for 5 min.
  6. Aspirate supernatant, gently resuspend pellet by flicking the tube, add 0.5 mL 10% FBS/DMEM, and pipette to mix.
  7. Take 10 µL for automated cell counting. Note: The counting solution contains 5 µL of dye and 5 µL of cell suspension, so divide the displayed cell number by 2.
  8. Adjust cell concentration to 5.0 × 105 cells/mL.
  9. Remove prepared collagen solution from wells.
  10. Seed 0.1 mL/well of the cell suspension into 96-well plates (3 wells per cell type).
  11. Incubate at 38.5°C with 5% CO2.

Cell Preparation 2

Use HEK293 cells:
  1. Aspirate 10% FBS/DMEM from flasks.
  2. Wash twice with PBS.
  3. Add 0.5 mL trypsin and incubate at 37°C for 5 min.
  4. Add 2 mL DMEM, pipette to resuspend, transfer to a 5 mL tube.
  5. Centrifuge at 900 rpm for 5 min.
  6. Aspirate supernatant, resuspend pellet in 0.5 mL 10% FBS/DMEM, pipette gently.
  7. Take 10 µL for automated counting (divide by 2 due to dye dilution).
  8. Adjust to 4.0 × 105 cells/mL.
  9. Remove collagen from wells and seed 0.1 mL/well.
  10. Incubate at 37°C with 5% CO2.

Viral Infection 1

Reagents volume(μl)
Dk/Hok/8/80(2.4*108PFU/ml) 380
BSA/DMEM 8740
total(1.0*107PFU/ml) 9120
  1. Wash cells once with BSA/DMEM.
  2. Dilute virus in BSA/DMEM to achieve 1 × 107 PFU/mL (5.0 × 104 cells/well × 10 MOI / 0.050 mL).
  3. Remove BSA/DMEM and add virus solution.
  4. Incubate at 38.5°C for 1 hour for viral adsorption.
  5. Remove virus solution and wash once with BSA/DMEM.
  6. Add BSA/DMEM and incubate at 38.5°C.

Viral Infection 2

Reagents volume(µl)
A/Victoria/361/2011(9.0*108PFU/ml) 56
BSA/DMEM 6244
total(8.0*106PFU/ml) 6300
  1. Wash cells once with BSA/DMEM.
  2. Dilute virus in BSA/DMEM to 8 × 106 PFU/mL (4.0 × 104 cells/well × 10 MOI / 0.050 mL).
  3. Remove BSA/DMEM, add 50 µL virus solution; mock wells receive 100 µL BSA/DMEM.
  4. Incubate at 37°C for 1 hour for adsorption.
  5. Remove virus solution, wash once with BSA/DMEM, then add BSA/DMEM and incubate at 37°C.

MDCK Cell Preparation for Titration

  1. Aspirate medium, wash twice with PBS.
  2. Cells were sequentially treated with trypsin to achieve complete detachment, with incubations of 2 mL (immediate removal), 2 mL (7 min + 5 min), 1 mL (5 min), and 1 mL (3 min).
  3. Add 5% FCS/DMEM, pipette to resuspend cells, and transfer to a tube.
  4. Centrifuge at 900 rpm for 5 min.
  5. Aspirate supernatant, resuspend in 10 mL 5% FCS/DMEM.
  6. Take 10 µL and transfer to a counting chamber for cell counting.
  7. Adjust to 2.5 × 105 cells/mL.
  8. Seed 100 µL/well into 96-well plates and incubate at 37°C with 5% CO2.

MDCK Viral Infection (Titration)

Use 9 hpi samples.
  1. Thaw 100 µL virus.
  2. Aspirate MDCK medium, wash twice with BSA/DMEM, incubate at 37°C.
  3. Add 100 µL TPCK trypsin to BSA/DMEM 75 mL.
  4. Add 135 µL/well to new 96-well plates.
  5. Perform 8-step 10-fold serial dilution by adding 15 µL virus per 3 wells per sample, transferring sequentially.
  6. Aspirate BSA/DMEM from MDCK cells, add 100 µL diluted virus per well.
  7. Incubate at 37°C with 5% CO2.

FACS Sample Preparation

Collect cells at 3, 6, 9, 12, and 14 hpi for analysis.
Reagents volume(µl)
PBS 100
Hoechst 0.1
FVD 0.1
Reagents volume(µl)
Annexin Binding Buffer 100
Annexin V-FITC 0.4
  1. Preparation of staining reagents.
  2. Aspirate culture medium and wash cells once with PBS.
  3. For pCK007, pCK008, pCK800, and Wild type at 9 and 12 hpi, collect supernatant for viral titration.
  4. Add trypsin and resuspend cells in 10% FBS/DMEM.
  5. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
  6. Add PBS staining solution containing Hoechst and FVD(+), vortex gently, and incubate 15 min at room temperature in the dark.
  7. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
  8. Add Annexin V solution, vortex gently, incubate 15 min at room temperature in the dark.
  9. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
  10. Add Annexin Binding Buffer, vortex gently.
  11. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
  12. Add 1% formaldehyde in Annexin Binding Buffer and fix cells on ice for 10 min in the dark.
  13. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
  14. Add Annexin Binding Buffer, vortex gently.
  15. Centrifuge at 1900 rpm for 5 min, aspirate supernatant.
  16. Add Annexin Binding Buffer, vortex gently.
  17. Store tubes in sample boxes wrapped in aluminum foil at 4°C.
Virus inactivation:
Two days post-collection, add 1% formaldehyde/Annexin Binding Buffer to inactivate virus. Centrifuge at 1900 rpm for 5 min, aspirate, and wash twice with Annexin Binding Buffer.

Fluorescence Observation

Observe MOCK, 6 hpi, 9 hpi, 12 hpi samples.
Reagents volume(µl)
Annexin Binding Buffer 100
PI 0.2
Annexin V-FITC 0.4
Hoechst 0.1
  1. Prepare staining solution.
  2. Wash cells once with PBS and add 100 µL staining solution per well.
  3. Incubate 20 min at room temperature in the dark.
  4. Observe under a fluorescence microscope.