Team:Jiangnan/Protocol

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1.IBRV detection by our fast detection method
(under patent protection)

(1)MDBK cells were cultured according to the conventional method. IBRV was added to cells when cell confluence reached 90%, and the supernatant was removed after cells completely exfoliated.
(2) The supernatant was put under 3 fold gradient dilution using 1640 medium, resulting in 5 gradients, numbered 1-5. Take 100 uL culture from each dilution to 500 uL centrifuge tube, and repeat this process for three times.
(3) The culture was boiled for 10 min, centrifuged at 10,000 RPM for 5 min. 15 uL supernatant was placed in a clean 200 uL centrifuge tube as the template for fluorescence quantitative PCR detection.

2.IBRV detection using qPCR in MDBK cell

(1) The specific qPCR primer was designed with gB gene of IBRV, and the product length was 108 bp. The qPCR primer sequence was as follows
siRNA IBRV gE-qPCR-F：CGTGGTGGTGCCAGTTAG
IBRV gE-qPCR-R：TCATCGTCGCTGTCGTCAT
(2) The SYBR-green fluorescence quantitative PCR reaction system of 10 uL was as follows:
5 uL SYBR Green PCR Master Mix
The upstream and downstream primers of each 0.4ul,
the template of different dilution degree 2 uL,
ddH2O complement to 10 uL
(3) Reaction conditions as follows:
95 ℃ modified 30 s,
95 ℃10 s，60 ℃30 s，72 ℃20 s，
40 cycles,
the conditions for dissolution curve analysis:
95 ℃ for 15 s, 30 s, 60 ℃ 95 ℃ for 15 s.

3.siRNA transfection
using Lipofectamine 2000

Use this brief procedure to transfect Stealth RNAi or siRNA into mammalian cells in a 24-well plate. All amounts and volumes are given on a per well basis. Use this procedure as a starting point; optimize transfections as described in Optimizing Stealth RNAi or siRNA Transfection.
(1) One day before transfection, plate cells in 500 µl of growth medium without antibiotics until they reach 30-50% confluent.
(2) For each transfection sample, prepare oligomer-Lipofectamine 2000 complexes as follows:
A. Dilute 20 pmol Stealth RNAi or siRNA oligomer in 50 µl Gibco™ Opti-MEM™ I Reduced Serum Medium without serum (resulting concentration of RNA in Opti-MEM is 400 nM). Mix gently
B. Mix Lipofectamine 2000 gently before use, then dilute 1 µl in 50 µl OptiMEM I Reduced Serum Medium. Mix gently and incubate for 5 minutes at room temperature. Note: Proceed to the next step within 25 minutes.
C. After the 5-minute incubation, combine the diluted oligomer with the diluted Lipofectamine 2000. Mix gently and incubate for 20 minutes at room temperature (solution may appear cloudy).

(3) Add the oligomer-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.
Incubate cells at 37°C in a CO2 incubator for 24-96 hours until you are ready to assay for gene knockdown. Medium may be changed after 4-6 hours.
Optimizing Stealth RNAi or siRNA Transfection
To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying RNA and Lipofectamine 2000 concentrations. Test 10-50 pmol RNA and 0.5-1.5 µl Lipofectamine 2000 for 24-well format. Depending on the nature of the target gene, transfecting cells at higher densities may also be considered when optimizing conditions.

Scaling Up or Down Transfections
To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine 2000, nucleic acid, cells, and medium used in proportion to the relative surface area, as shown in the table. With automated, high-throughput systems, a mixture volume of 50 µl is recommended for transfections in 96-well plates.
Note: You may perform rapid 96-well plate transfections by plating cells directly into the transfection mixture. Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100 µl volume. Cells will adhere as usual in the presence of complexes.

Figure 1. Several examples of different volume complexes

1Surface areas may vary depending on the manufacturer
2Volumes of dilution medium in Step 2a & 2b of DNA or RNA or RNAi transfection protocols

4.Plasma

1 ml blank DMEM medium was added to the 12-hole plate and placed directly under the low temperature atmospheric pressure plasma generator. The distance between the end of plasma beam and the medium is 1 cm, the injection port is vertically aligned to the center of the hole in the 12-hole plate, the controlled helium flow rate is 1 slm / min, and the output voltage of the high frequency power supply controlled by the transformer is 0.96-1.24kv. Plasma activated medium (PAM) could be obtained by repeating the aforementioned process. When cells were incubated with the viruses, the active oxygen and the active nitrogen in the culture medium had a beneficial effect on virus replication. 500000 MDBK cells were placed in the 6-hole plate for 24 hours. Then the original culture medium was changed to 1 ml PAM. IBRV were then added and incubated with MDBK cells for 1 hour before changing the medium to the maintaining 1640 medium (supplemented with serum at 2% final concentration). The culture was collected for virus titer detection after cell lysis.

Figure 2. Protocol of cold atmospheric plasma activated medium preparation.

1. Digestion

(1) Add DNA into an empty centrifuge tube (1ug).
(2) Add 5 ul Green Buffer.
(3) Add Enzyme A and Enzyme B, each for 2 ul.
(4) Add ddH2O to 20 ul volume.
(5) Incubate the reaction system at 37 ° C for 1 h.

2. Ligation

(1) Add 1 ul DNA into an empty 1.5 ml microcentrifuge tube (1ug).
(2) Add 4 ul target sequence (1ug).
(3) Add 5 ul Ligation Mix.
(6) Ligate at 16° C for 1 h.
(7) Transform the ligation product into competent E. coli.
(8) Incubate at 37 ° C for 1 h.

3. Isolation of plasmid

(1) Take the LB culture liquid (2 ul) and plate it on an Amp-resistant LB plate.
(2) Culture at 37 ° C for 12 to 16 h.
(3) Pick out the monoclonal grown on the plate and culture in LB medium for 12 h
(4) Colonal PCR verification.

Tablet 1. Colonal PCR verification Reaction system.

4. Lentivirus transfection

Day 1 Cell seeding: Seed the logarithmic growth phase cells in a 6-well plate and cultivate them until 70% -90% confluence after 24 h.
Day 2 Infection:
(1) Use ENi.S to dilute the lentivirus to 1×107 TU/mL, naming the solution ‘A’. Prepare the ENi.S solution with Polybrene (50 mg/L), naming the solution B. Then mix the solution ‘A’ and B in a ratio of 1:9 by volume.
(2) Pipette the supernatant of the 6-well plate and add ‘AB’ mixture (1.5 mL) into each well, and put the cells in an incubator cultivation.
(3) After infection for 12h, change the cells back to conventional medium.
Day 3-4 Cell culture: Change cell medium when cells grow to 90% confluence.
Day 5 Test: Observe GFP expression in cells using fluorescence microscopy.

5. Liposomal transfection

Day 1 Cell seeding: Seed the logarithmic growth phase cells in a 6-well plate and cultivate them until 70% -90% confluence after 24 h.
Day 2 Transfection:
(1) Mix Lipofectamine 3000(3.75 ul) with fresh medium (100 ul), naming the solution ‘A’. Mix plasmid (2.5 ug) and P3000(5 ul) with fresh medium (100 ul), naming the solution ‘B’.
(2) Mix the solution ‘A’ and ‘B’ together and incubate at room temperature for 15 min.
(3) Pipette the supernatant from the 6-well plate and then add ‘AB’ liquid.
(4) After incubation for 6-8 h, replace it with complete medium.
Day 3-4 Cell culture: Change cell medium when cells grow to 90% confluence.
Day 5 Test: Observe GFP expression in cells using fluorescence microscopy.

6. Electroporation transfection

Day 1 Material preparation: Prepare materials for electrorotation, such as cells (2×106 cells/mL) and plasmid (20 ug) with concentration greater than 1 ug/ul for each sample.
Day 2 Transfection:
(1) Wash and resuspend cells with pre-chilled PBS after trypsinization.
(2) After centrifugalization for 5 min, add PBS (200 ul) to resuspend cells (2×106cells/mL) at room temperature, and then add plasmid (20 ug), salmon sperm DNA (10 ug) together.
(3) Place the solution in a pre-chilled shock cup (2 mm) which is sterilized with ethanol in ice bath for 1 min.
(4) Give the shock cup shocks for three times with the cell electroporation apparatus (350 V, 500μs) and each interval is 1min.
(5) Wash the shock cup with DMEM medium containing 10% FBS and transfer cells to a 6-well plate for a final volume of 2 mL/well.
Day 3 Medium changing: Remove the supernatant from the 6-well plate and replace it with fresh medium.
Day 5 Test: Observe GFP expression in cells using fluorescence microscopy.

7. Calcium phosphate transfection

Day 1 Material preparation:
(1) Seed the logarithmic growth phase cells in a 6-well plate and cultivate them for 24 h when they reach 50% -70% confluence.
(2) Precipitate the plasmid using ethanol and the solution concentration is 500 ng/ul. Dry the precipitate in the air.
Day 2 Transfection:
(1) Change the medium 4 h before transfection. Add CaCl2 (9 ul 2 mol/L) and DNA (5 ug) to ddH2O (100 ul), and mix them up to gain the solution ‘A’. Configure the solution ‘B’ with HBS (100 ul 2×).
(2) Add the solution ‘A’ into the solution ‘B’ drop by drop, and use another pipette to blow the solution. This process should be kept slowly for about 1-2 min. Finally, add the ‘AB’ solution to a 6-well plate and place the 6-well plate in the incubator overnight.
Day 3 Medium changing: Remove the supernatant from the 6-well plate and wash cells twice using D-PBS, and replace it using fresh medium.
Day 5 Test: Observe GFP expression in cells with fluorescence microscopy.

8. Optimum antibiotics screen concentration assay

Day 1 Cell seeding: Seed the logarithmic growth phase cells in 96-well plates for 100 ul per well and cultivate them for 24 h when they reach 50%-70% confluence.
Day 2 Dosing: Set nine groups and three parallel wells in each group. Add antibiotics G418 to reach different concentration gradients in each group, i.e., 0, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4 mg/mL.
Change the fluid every three days and add antibiotics G418 at the same time. When cells die after 7-10 days of dosing, that dosage is considered the optimum screen concentration.

9. Genetically modified cell strain selection

(1) Electroporation transfection
(2) Steady transplanted Cell strain screening
①After transfection for 48 h, change the medium and add antibiotic G418 (dissolved in HEPES) to a final concentration of 2 g/L. When the number of cell deaths increases, add antibiotics to the medium again.
②After one week of antibiotic screen, select monoclonal cells and expand them using antibiotic G418(1 g/L) and finally obtain the genetically modified cell strain.
(3) Cell cryopreservation
①After a large number of cells were cultured, trypsinize and centrifuge the cells, and prepare cells to a cell suspension (1×106 cells/mL) with the cell cryopreservation solution.
②Dispense cells into cell cryopreservation tubes for 1 mL each with name, generation, date and operator marked on.
③Put cryopreservation tubes in a cryopreservation box, and freeze it in a refrigerator at - 80° C overnight. Transfer to a liquid nitrogen tank the next day for conservation.
Cell cryopreservation formulation: 50% cell culture medium, 40% serum, 10% DMSO.

10. RNA extraction

(1) Collect cells by centrifugalization for 5 min. Plus 300-400ul of Trizol every 100,000 to 10 million cells.
(2) After adding 1 mL Trizol, repeatedly blow the sample until it is fully lysed. Place it at room temperature for 5 min, and the protein nucleic acid complex is completely separated.
(3) After adding 200 ul chloroform, shake it hard for 15 s, and leave it at room temperature for 2 min.
(4) Centrifuge (4 ° C, 12000 rpm) the sample for 10 min. Now, the sample is divided into three layers: red organic phase, intermediate layer (protein) and the upper layer of colorless aqueous phase. RNA is mainly in the upper aqueous phase.
(5) Remove the upper aqueous phase to a new RNase-free centrifuge tube. Add an equal volume of absolute ethanol to the aqueous phase and mix by inversion.
(6) Add the obtained liquid to the adsorption column. If it cannot be added at one time, it can be divided for several times. Centrifuge the sample at 12000 rpm for 20 s, and the waste liquid is drained.
(7) Add 700ul Buffer RW1 to the adsorption column. Centrifuge at 12000rpm for 20 s, and then drain the waste liquid.
(8) Add 500ul Buffer RW2 to the adsorption column. Centrifuge at 12000rpm for 20 s, and then drain the waste liquid.
(9) Repeat step 8.
(10) Centrifuge the vacant tube at 12000 rpm for 2 min.
(11) Pour off the waste liquid, and then place the adsorption column open at room temperature for 5 min.
(12) Place the adsorption column in a new centrifuge tube, and then add 30 ul of RNase-free water to the middle of the adsorption column. After leaving it at room temperature for 1 min, centrifuge it at 12000 rpm for 1 min, and collect the RNA solution.
(13) Measure RNA concentration. Test the product using agarose gel electrophoresis.
Reagents:

Table 2. Agarose gel electrophoresis Reaction system.

11. RT-PCR

(1) Primers of the RT-PCR design.

Table 3. Primers in this study.

(2) Seed MDBK cells (negative control) and MDBK-N4 cells on six-well plates and cultivate them until 80%-90% confluence.
(3) Extract the total RNA of cells.
(4) Add the reagents in table 3 into a centrifuge tube. Place the tube at 37° C for 15 min and then give it a heat hit at 85° C for 5 s.

Table 4. Reverse transcribe reaction system.

(5) Using cDNA as a template, amplify the Nectin 4 and GADPH with reverse transcription PCR primers by PCR.

Table 5. cDNA amplification reaction system.

(6) Test the PCR amplification products using agarose gel electrophoresis and observe and analyze it using a gel imager.

12. Western blot

(1) Seed MDBK (negative control) and MDBK-N4 cells in six-well plates and cultivate then until 80%-90% confluence.
(2) Wash the cell sample with pre-cooling PBS. Add 1% PMSF and membrane protein lysate (100ul) to the lysate. Cleave 50-100 million cells for 10 s on ice. Flick and then centrifuge the sample for 5 min (10000rpm, 4°). Carefully pipette the supernatant.
(3) Mix cell sample (40 ul) with loading buffer (10 ul) and then boil the solution for 5 min.
(4) Take the protein sample (30 ug) to perform polyacrylamide gel electrophoresis (150V 90min).
(5) Cut off the concentrated gel and bromophenol blue at the bottom. Based on the size of the cut gel, cut a larger PVDF membrane and six larger filter papers. Following that, align the membrane with the center of the glue and place it in the middle of the filter paper to form ‘3 layers of filter paper + film + glue + 3 layers of filter paper’. Transfer the membrane at a constant voltage of 15 V for 12 min.
(6) Wash the membrane three times with TBST. Each time place it on a shaker for 10 min. Add with 5% skim milk powder into the TBST solution to prepare a blocking solution. Block the membrane at room temperature for 60 min.
(7) Wash the membrane three more times with TBST. Each time place it on a shaker for 10 min. Place it in a primary antibody diluted with GAPDH (1:2500) and ALDH (1:500), and incubate at room temperature for 90 min.
(8) Wash the membrane three more times with TBST. Each time place it on a shaker for 10 min. Add the HRP-labeled secondary antibody diluted with GAPDH (1:3000) and ALDH (1:2000), and incubate at room temperature for 60 min.
(9) Wash the membrane three times with TBST. Each time place it on a shaker for 10 min. Mix liquid ‘A’ (700ul) and liquid ‘B’ (700ul) away from the light. Apply it to the membrane. Observe that Bcl reagent developed color by using a chemiluminescence gel imager.

13. Indirect Immunofluorescence

(1) Digest logarithmic growth phase cells with 0.25% trypsin to prepare a single cell suspension.
(2) Inoculate the cells into a 24-well plate and incubate them in a 5% CO2 incubator for 1 day until the cells are nearly grown into a single layer.
(3) Add 4% paraformaldehyde into the 24-well plate, and fix cells for 30 min at 37° C.
(4) Add the PBS solution which contains 1% Triton X-l00 into the 24-well plate, and incubate for 30 min at 37° C to increase the permeability of the cell membrane.
(5) Block cells with non-immune animal serum at 37 ° C for 40 min. After pipetting the serum, label the wells and add the antibody (1:100), and keep in the wet box at 4 ° C overnight.
(6) Wash cells with pre-cooling PBS solution for three times and each time lasts for 5 min. Add the corresponding fluorescently labeled secondary antibody (1:50) in the dark. Following that, add fluorescein isothiocyanate (FITC) (green) labeled secondary antibody (1:50) and place in the wet box at 37 ° C for 30 min. Wash cells with pre-cooling PBS solution for three times and each time lasts for 5 min. (7) Counterstain cell nucleus with 4',6-diamidino-2-phenylindole (DAPI) (1:1000) in the dark at 37 ° C for 20 min.
(8) Wash cells with pre-cooling PBS solution for three times and each time lasts for 5 min. Observe the imaging with inverted fluorescence microscope (TE2000, NIKON).
The negative control uses PBS solution instead of the primary antibody, and remaining steps are the same as those in the experimental group.

1. Cell batches

(1) Pour out the medium in T25 flasks.
(2) Draw PBS to wash T25 flasks.
(3) Digest cells using 500ul trypsin.
(4) Store the cells in a thermostat for 1 minute at 37 degree Celsius.
(5) Add 2ml fresh medium in T25 flasks to wash repeatedly.
(6) Put 1/3 volume liquid into a new T25 flask and fill it with 5ml new medium. The serum level of medium is 10%.

2. Cell recovery

(1) Remove the cryopreserved cells from liquid nitrogen and quickly put them into a water bath to melt cells.
(2) Add melted cells to the prepared medium (10 ml or so) in a 15 ml centrifugal tube.
(3) Centrifuge cells and remove the supernatants.
(4) Add new medium and fully mix cells.
(5) Transfer cells from centrifuge tubes to T25 flasks.

3. Cell seeding

(1) Pour out medium in T25 flasks.
(2) Wash cells with PBS and rinse 2-3 times.
(3) Digest cells with 500ul trypsin.
(4) Store cells in a thermostat at 37 degree Celsius for 1 minute (BHK-21 CHO-K1) or 3minutes(MDBK).
(5) Add 2ml new medium.
(6) Cell counting using a small fraction of cells (We should make sure the number of cells reaches 200000/ml in 6-well plate). Then add 2ml medium to each hole.

4. RNA extraction

(1) The cells are collected by centrifugation, add 1ml Trizol to break up cells. Place the cells at room temperature for 5 minutes to completely crack the sample.
(2) Add 200ul chloroform and shake violently for 15 seconds, then place it at room temperature for 2minutes.
(3) After 4 degree Celsius, 12000 rpm centrifugation for 10 minutes, the samples are divided into three layers: the red organic phase, the middle layer (proteins) and the upper clear water phase. RNA is mainly in the upper water phase, move the upper water phase into a new RNase-free centrifuge tube.
(4) Add equal volume of anhydrous ethanol in the obtained water phase, and mix the liquid upside down.
(5) Add the obtained liquid to adsorption columns and centrifuge for 20 seconds at 12000rpm to remove the waste liquid
(6) Add 700ulBuffer RW1, 12000rpm to the adsorption column, centrifuge for 20 seconds and discard the waste liquid
(7) Add 500ulBuffer RW2, 12000rpm to the adsorption column, centrifuge for 20 seconds and discard the waste liquid
(8) Repeat step 7
(9) Centrifuge at 12000rpm for 2min, pour the waste liquid, and the adsorption column is exposed to room temperature for 5minutes.
(10) Place the adsorption column in a new centrifugal tube, 30ul RNase-free water is added to the middle of the column. Place the centrifugal tube at room temperature for 1 minute, centrifuge for 1 minute at 12000 rpm, and then collect RNA solution.

5. siRNA transfection(6-well plate)

(1) Cell seeding: The convergence degree reaches 30%-50% after the cells are cultured on 6-well plates for 24 hours.
(2) Transfection: In order to ensure the test results, this procedure requires the use of barrels and centrifuge tubes without RNase. ‘A’ solution is prepared by mixing 3ul Lipofectamine 3000 with 100ul fresh medium, and then adding 25-100nmol siRNA into 100ul fresh medium to prepare ‘B’ solution. ‘AB’ solution is mixed and incubated at room temperature for 1 minute. The 6-well plate is discarded for supernatant, and the cells are incubated overnight by adding 900ul in ‘AB’ solution.

Table The sequence of siRNA used in this study

6. Reverse transcription

(1) Prepare the removal of genomic DNA reaction fluid ‘A’:

Store fluid ‘A’ 5min at room temperature or 2min at 42 degree Celsius.

(2) Prepare the reverse reaction solution ‘B’：

Mix ‘A’ and ‘B’ solutions, place the PCR Tube at 37 degree Celsius for 15 minutes, then place it at 85 degree Celsius for 5 seconds, the complete cDNA is obtained and stored at –80 degree Celsius.

7. qPCR(Quantitative real-time PCR)

(1) The cDNA is diluted to the final concentration of 10ng/ul.
Melt SYBR Mix at room temperature.
(2) Prepare PCR reaction mixture according to Table 1.
(3) Add 8ul PCR reaction mixture to a 96-well plate according to Table 2.
(4) Add 2ul template cDNA solution.
(5) Seal the 96-well plate with transparent adhesive film
(6) After mixing with vortex, centrifugally collect the solution to the bottom of the tube.
(7) Run PCR reaction according to Table 3.

Table 1. PCR reaction mixture formulation

Table 2. PCR reaction layout of the 96-cell plate

Table 3. PCR procedure

8. Cell count

(1) Place the coverslip over the hemocytometer counting chamber and using a Pasteur pipette, place a drop of the cell suspension at the edge of the “V” shape of the chamber. Allow the suspension to be drawn into the chamber by capillary action. Care should be taken not to overfill or underfill the chamber. Fill the opposite chamber in the same manner.
(2) Place the chamber on the microscope stage.
(3) The hemocytometer consists of nine 1 mm squares divided into smaller squares. One of the 1 mm squares represents a volume of 0.1 mm3 or 10-4 ml. Using the 10objective, count the number of cells in a 1 mm square area. If there are fewer than 100 cells in a square mm, 2 or more 1-mm square areas should be counted and the results averaged.
(4) Use the same procedure to count cells on the other side of the hemocytometer.
(5) To calculate the concentration of cells, first calculate the average of all 1mm2 areas counted and apply this formula: c=n/v where: c = cell concentration in cells/ml, n = avg. number of cells/mm2 area v = volume counted = 10-4
Thus: c = n x 10-4