Cloning of target protein Panobody to pET-24d(+) vector

Materials required:

pET-24d(+) vector, XhoI, HindIII, T4 Ligase, Top 10 cells, BL21(DE3) cells, 10X NEBuffer, Wizard® SV Gel and PCR Clean-Up System, TIANprep Mini Plasmid Kit, Buffer for Nickel column purification, Milli-Q water, 20% ethanol, HiTrap™ chelating HP column
Expression vector: pET-24d(+)
Plasmid from Genscript: pUC-Panobody

Schedule:

1. Restriction digestion of pET-24d(+) vector and pUC-Panobody vector with XhoI and HindIII to generate compatible ends
2. DNA gel electrophoresis of restriction digested products
3. Gel purification of restriction digested products
4. Ligation of Panobody gene and pET-24d(+)
5. Transformation of ligation product into TOP10 competent cells
6. Colonies picking and inoculation
7. Recombinant plasmid purification
8. Sequencing
9. Transformation of selected plasmids into BL21(DE3) competent cells
10. Colonies picking and inoculation
11. Protein expression
12. Polyacrylamide Gel Electrophoresis analysis to check expression level
13. Protein purification by Ni-NTA spin column
14. Polyacrylamide Gel Electrophoresis analysis to check purity
15. Liquid Chromatography Mass spectrometry analysis

Restriction digestion of pET-24d(+) vector and pUC- Panobody vector with XhoI and HindIII to generate compatible ends

1. Set up the restriction digestion reaction as follow:

   pET-24d(+) vector / pUC-Panobody vector	1 µg
   XhoI (10 units/ µL)	0.5 µL
   HindIII (10 units/ µL)	0.5 µL
   10X NEBuffer	5 µL
   ddH2O	34 µL
   Total volume	50 µL

2. Incubate at 37°C for three hours.
3. Inactivate the enzymes by incubating at 65°C for 20 minutes.

DNA gel electrophoresis of restriction digested vector

1. Place the pre-prepared 0.8% (w/v) agarose gel containing Gelred (5 µl of Gelred to 50 ml of agarose gel) into the gel tank.
2. Fill the gel running tank with the gel running buffer (1X TAE) until it just covers the gel surface.
3. Spin down briefly (10 seconds) the double restriction digested cloning vector tube.
4. Add 10 µl of loading dye (6X) to the digestion reaction. Mix gently and spin down.
5. Carefully load ALL the reaction mixture into the wells of the gel.
6. Load 5 µL of the 1 kb DNA size marker to a lane.
7. Place the lid onto the gel tank. Connect the electrodes to the power supply.
8. Perform electrophoresis at about 150V (constant voltage) for around 30 minutes.
9. Run until the dye front has moved about half of the way down the gel.
10. Switch off power.
11. Carefully lift the gel tray and place the gel onto a UV light box.
12. Make sure the long UV wavelength is selected.
13. Make sure the UV shield is properly placed to block UV from your eyes and skin.
14. Switch on the UV light box.
15. Observe the gel pattern and then cut out the gel pieces that contain the DNA fragments you want.

Gel purification of restriction digested products

1. Measure the weight of 1.5 mL microcentrifuge tubes. Label sample name and weight of tube clearly on each tube.
2. With a pair of cover slip, cut out the slice(s) of agarose gel containing the DNA band(s) of interest (pET24d: ~5.3 kb, Panobody: 860bp).
3. Calculate the net weight of agarose.
4. Follow the DNA extraction procedure below:

Dissolving the Gel Slice

1. Add 10 μl Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50–65°C until gel slice is completely dissolved.

Binding of DNA

1. Insert SV Minicolumn into Collection Tube. Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute.
2. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

Washing

1. Add 700 μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
2. Repeat Step 3 with 500 μl Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
3. Empty the Collection Tube and recentrifuge the column assembly for 1 minute.

Elution

1. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube. Do not let the Minicolumn touch the solution inside the Collection Tube.
2. Add 30 μl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
3. Discard Minicolumn and store DNA at -20 °C.

Ligation of Panobody gene and pET-24d(+)

1. Before ligation, measure the concentrations of the gene insert and the cloning vector eluates.
2. Setup the ligation reactions as follow:

   Tube number (molar ratio of vector to insert)	1:3
   Add milli-Q H2O to a final volume of 9.5 µL
   Ligation buffer 10X	1 µL
   Double restriction digested cloning vector	50 ng
   Double restriction digested insert Panobody	28 ng
   Then adding each tube
   T4 DNA Ligase	0.5 µL

3. Make sure you add all the components to the bottom of the tube in the following order:
   1. Water
   2. Buffer
   3. DNA
   4. Enzyme
4. Mix briefly and gently.
5. Spin down all contents to the bottom of the tube.
6. Incubate at room temperature (25 °C) for 3 hours.
7. Store at -20 °C until perform transformation.

Transformation of ligation product into TOP10 competent cells

1. Set up 2 transformation tubes as follow:

   Tube number		Amount
   1	Ligation reaction tube	10 µL
   vec	Vector plasmid (+ve control)	1 µL

2. Thaw the competent cells on ice. Keep them on ice whenever its possible before the heat shock.
3. Add 10 μL of the ligation reaction mixture and 1 µL vector to each 100 µL of the competent cell suspension.
4. Mix the contents of the tubes by stirring gently with the pipette tips and store on ice for at least 10 minutes.
5. Transfer the tube to a 42 °C water bath for 45 seconds.
6. Immediately transfer the tube to ice bath.
7. Allow the cells to chill for 2 minutes.
8. Add 900 µl of LB medium to the tube.
9. Use tape to secure tube cap, incubate at 37 °C with shaking for 45 minutes.
10. Centrifuge the cells at 7000 rpm for 5 minutes. Discard most of the supernatant but remain ~100 μL inside. Resuspend the cells using pipette.
11. Add all 100 µL of cell suspension onto the agar plates, respectively.
12. Use sterile spreader to spread them out evenly on the agar plate.
13. Let it adsorb into the agar about 5 minutes.
14. Turn the plate over and incubate the plates at 37 °C overnight.

Colonies picking and inoculation

1. Prepare 2 tubes each 5ml of LB broth. Pick two single colonies from the ligation plate and inoculate each colony in one tube of LB broth. Incubate overnight at 37 °C with shaking.

Recombinant plasmid purification

Purify the recombinant plasmids from the liquid cultures according to the following:

1. Harvest the bacterial cells by centrifugation at 7,000 rpm in a bench-top centrifuge for 5 min (or 4,000 rpm for 15 min) at room temperature, remove supernatant.
2. Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube.
3. Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.
4. Transfer the resuspended cell to a new 1.5ml microcentrifuge tube. Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times. Note: Do not vortex!! If necessary, continue inverting the tube until the solution becomes viscous and slightly clear, but less than 5 min.
5. Add 350 μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000rpm (~13,400 × g) in a table-top microcentrifuge.
6. Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.
7. Wash the Spin Column CP3 by adding 600 μl Buffer PW (ethanol (96%-100%) has been added) and centrifuge for 60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube. Repeat Step 7 one more time.
8. Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW.
9. Place the Spin Column CP3 in a clean labeled 1.5 ml microcentrifuge tube to elute DNA, add 30μl Buffer EB to the center of the Spin Column CP3 incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
10. Separate the 10μl sample into a new clean labeled 1.5 ml microcentrifuge tube and add 10μl Double Distilled Water (DDW) into the tube.
11. Send the microcentrifuge tube containing DDW to the sequencing company and check the sequencing result.
12. Test the concentration and OD reading for the left sample.
13. Send the samples to sequencing company to check for the presence of insert.

Transformation of selected plasmids into BL21(DE3) competent cells

1. Use 1 tube of competent Bl21(DE3) E. coli cells (100 µl) for each plasmid.
2. Add the required plasmid to the cells and mix by flicking the tube.
3. Chill in ice for exactly 10 minutes.
4. Transfer to 42 ºC for exactly 45 seconds.
5. Return to ice for exactly 2 minutes.
6. Add 900 µL of LB (without antibiotics) to make the solution up to ~1000 µL.
7. Incubate at 37 °C for 45 minutes with shaking (~150 rpm).
8. Spread 100 µL of culture to the plate. Choose the nutrient agar plate with correct antibiotics.
9. Leave the plates to dry.
10. Invert plates and incubate at 37 ºC for overnight.

Colonies picking

1. Pick 2 colonies from the plate and transfer each to 5 ml LB medium (with antibiotic added).
2. Incubate overnight at 37 ℃ with shaking.

Protein expression

(for small scale expression)

1. Inoculate 0.2 ml of overnight starter culture into 10 ml sterile LB medium containing antibiotics.
2. Incubate at 37 °C with shaking (~250 rpm).
3. Setting the BLANK of spectrophotometer using the LB medium. Every 30 minutes, take out the broth culture from the shaker and aseptically transfer 0.1 ml to a clean cuvette containing 0.9 ml LB. Pipette up and down to mix well. Avoid bubbles. Determine its O.D. Put the 10ml culture back into the shaker at 37 °C and keep shaking until O.D.600 reaches 0.06 (after dilution).
4. Separate the culture into two falcon tubes (each one has 5 ml now). Name one tube as “uninduced (UI)” and another tube as “induced (I)”.
5. Immediately transfer the tube to ice bath.
6. Allow the cells to chill for 2 minutes.
7. Add 900 µl of LB medium to the tube.
8. Induce with 0.5 mM IPTG (stock concentration: 100 mM) in the “(I)” tube.
9. Express protein for overnight at 16 °C /25 °C /30 °C. Aliquot samples at different times (6 hours, 24 hours) for gel electrophoresis.
10. Centrifuge the cells at 7000 rpm for 15 minutes at 4 °C. Discard supernatant. Store the cell pellets at -20 °C for overnight.
    Conditions

    Temp	6h	24h
    16°C
    25°C
    30°C

Protein analysis: SDS-PAGE

Preparation of the gel

1. Use 70% ethanol and Kimwipes to clean both long and short glass plates.
2. Assemble the Mini-PROTEIN 3 Casting Frame and Casting Stand.

   

3. Casting the resolving gel:
   1. Place a comb completely into the assembled gel sandwich. Mark on the glass plate with a marker pen at 1 cm beneath the teeth of the comb. This will be the level to which resolver solution is poured. Remove the comb for later use in the casting of stacking gel.
   2. Mix the below reagents in a 5ml tube:

      Resolver A	2 ml
      Resolver B	2 ml
      10% APS	20 ul
      TEMED	2 ul
      Resolver Solution	~4 ml

   3. APS and 10% TEMED should be added last.
   4. Use 1 mL pipette to transfer the resolving gel into the gel chamber up to the marking made above. Avoid introduction of air bubbles.
4. Casting the stacking gel:
   1. Mix the below reagents in a 5ml tube:

      Stacker A	2 ml
      Stacker B	1 ml
      10% APS	10 ul
      TEMED	2 ul
      Resolver Solution	~2 ml

   2. 10% APS and 10% TEMED should be added last.
   3. Use 1 mL pipette to transfer the stacking gel above the resolving gel as soon as possible. Avoid introduction of air bubbles.
   4. Insert the comb into the space between the 2 glass plates.
   5. Allow the gel to polymerize for 30 min.
   6. After polymerization, cover the gel by wet tissue paper and keep inside a seal bag at 4°C for storage.

Cell lysis by sonication

Extracting Protein from Bacteria

We will use B-PER® Bacterial Protein Extraction Reagent to extract soluble proteins.

1. Add 0.2 mL of B-PER Reagent to the cell pellet from 5 ml culture. Pipette the suspension up and down until it is homogeneous.
2. Transfer the lysate to a 2 mL microcentrifuge tube.
3. Pipette 15 μl of lysate to a new 1.5 mL microcentrifuge tube. It will be used for SDS-PAGE analysis.
4. Incubate the remaining lysate for 15 minutes at room temperature.
5. Centrifuge the lysate at 13000 rpm for 10 minutes to separate soluble proteins from insoluble proteins.
6. Transfer the soluble protein carefully to a new 1.5 mL microcentrifuge tube. It will be used in protein purification.

SDS-PAGE gel analysis

Purification of protein by Ni-NTA Affinity Chromatography

Materials required:
Equilibration buffer: 20 mM sodium phosphate buffer with 0.5 M NaCl, pH 7.4
Elution buffer: 0.2 M imidazole added to equilibration buffer, pH 7.4
Cleaning buffer: 0.5 M imidazole added to equilibration buffer, pH 7.4
DDI water
20% ethanol
5 ml HiTrap™ chelating HP column
1.5 ml-microcentrifuge tubes
Bench-top microcentrifuge
Pipette and pipette tips
Procedure:

1. Before loading the sample onto the Nickel affinity column, the sample was filtered through 0.45 µm and 0.2 µm filters.
2. The column used was a 5 ml HiTrap™ chelating HP column from Cytiva. The column was first injected with 0.1 M Nickel (II) sulfate and washed with Milli-Q water to remove unbound Nickel ions.
3. Then it was pre-equilibrated with the starting buffer (0.5 M sodium chloride, 0.02 M sodium phosphate, pH 7.4).
4. The sample was loaded onto the column, and the column was washed with 10 column volumes (CV) of starting buffer to remove unbound proteins.
5. Washing was performed with 10% imidazole concentration. Elution was performed with 30% and 100% imidazole concentrations. Fractions showing positive UV absorbance at 280 nm were analyzed by SDS-PAGE to check for purity.
6. The fractions containing the target protein were pooled, and buffer exchange to PBS was carried out using an Amicon® Ultra with a 10 kDa pore size.

Buffer exchange

Materials required:
10kDa Amicon Ultra centrifugal filter units
PBS
1.5 mL microcentrifuge tube
Procedure:

1. Add protein solution (in elution buffer) to the filter unit. Using the PBS to fill up the filter.
2. Insert the filter unit into the centrifuge and balance. Centrifuge the filter at 5000 rpm until the solution gets to 0.5 mL in the filter.
3. After the initial spin, discard the filtrate. Add PBS to the concentrated protein in the filter device. Centrifuge again under the same conditions as before. Repeat this wash step several times (6-8 times), each time discarding the filtrate and adding fresh PBS.
4. After the last centrifugation, use the pipette to collect the protein and transfer it into a 1.5 mL microcentrifuge tube. Remember to pipette up and down to collect all protein.
5. Add an equal volume of PBS into the filter to wash the rest of the protein and collect it into the same 1.5 mL microcentrifuge tube.

Bradford Assay

Materials required:
Bradford reagent
Protein standards (BSA: 1 mg/mL)
Double Distilled Water (DDW)
Protein
96 wells plate
Procedure:

1. Use the standard protein BSA and DDW to create a calibration curve and dilute protein.

   BSA			Protein Sample
   10 μl BSA	10 μl BSA	10 μl BSA	1 μl protein + 9 μl DDW	1 μl protein + 9 μl DDW	1 μl protein + 9 μl DDW
   8 μl BSA + 2 μl DDW	8 μl BSA + 2 μl DDW	8 μl BSA + 2 μl DDW	0.5 μl protein + 9.5 μl DDW	0.5 μl protein + 9.5 μl DDW	0.5 μl protein + 9.5 μl DDW
   6 μl BSA + 4 μl DDW	6 μl BSA + 4 μl DDW	6 μl BSA + 4 μl DDW
   4 μl BSA + 6 μl DDW	4 μl BSA + 6 μl DDW	4 μl BSA + 6 μl DDW
   2 μl BSA + 8 μl DDW	2 μl BSA + 8 μl DDW	2 μl BSA + 8 μl DDW
   10 μl DDW	10 μl DDW	10 μl DDW

2. Dilute Bradford reagent with DDW (Bradford: DDW=1:3) and add 100 μl Bradford reagent to each well with a multichannel pipette. Wait for 15 mins at room temperature.
3. Measure the absorbance at 595 nm using a spectrophotometer.
4. Plot the absorbance of the standards against their respective concentrations to create a standard curve.
5. Use the standard curve to determine the concentration of the protein by interpolating the absorbance values of your unknown samples.

Molecular Mass analysis by LC-ESIMS

1. Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-ESIMS) experiments were performed with an Agilent 6540 QTOF mass spectrometer coupled with an Agilent 1290 Infinity UHPLC system.
2. A purified Nanobody protein sample was injected into a C4 LC column and eluted with a linear gradient from 95% solvent A:5% solvent B to 5% solvent A:95% solvent B, where solvent A is milliQ water and solvent B is acetonitrile, each with 0.1% formic acid.
3. The mass spectrometer was operated in positive ion mode. ESIMS data was acquired with m/z range of 600-2000, from which multiply-charged mass spectrum was obtained. The multiply charged mass spectrum was deconvoluted by the MassHunter BioConfirm program to obtain the molecular mass of proteins.

Materials

• 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder (Sigma-Aldrich 475989)
• 96-well cell culture plates
• Gemcitabine (Macklin G824361)
• Culture grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich D2650)
• Phosphate-buffered saline (Solarbio P1000)
• Fetal Bovine Serum (FBS) (GibcoTM A5256701)
• Panobody (Nanobody)
• Dulbecco’s Modified Eagle’s Medium (DMEM), high glucose (Gibco™ 12100061)
• Trypsin-EDTA (0.25%) (Gibco™ 25200072)
• Penicillin-Streptomycin (10,000 U/mL) (Gibco™ 15140122)
• Trypan Blue Solution, 0.4% (Gibco™ 15250061)

Preparation of reagents

PBS (1X)

1. Dissolve 5 PBS tablets in 500 mL dH₂O
2. Autoclave before use.

0.05% Trypsin with 0.05% EDTA

1. Thaw the 0.25% Trypsin at room temperature.
2. Dissolve 50 mL Trypsin solution in 200 mL 1X PBS.
3. Aliquot to 40 mL per tube with the use of 50 mL tubes.

MTT stock solution (5 mg/mL)

1. Prepare a 5 mg/mL MTT stock solution by dissolving 50 mg of MTT powder in 10 mL of 1X PBS.
2. Vortex until the powder completely dissolved.
3. Aliquot to 1mL with the use of 1.5 mL microcentrifuge tubes.
4. MTT solution is light sensitive. Foil the aliquots and store in darkness at -20℃.

Gemcitabine stock solution (200 mM)

1. Dissolve 52.62 mg of Gemcitabine powder into 1000 μL of DMSO to create a 200 mM stock solution.
2. Subsequent concentrations are prepared from the 200 mM stock solution with DMSO.

PANC-1 Monolayer Cell Culture

1. Mock PANC-1 are cultured in DMEM supplemented with 10% FBS and 1% PS at 37°C incubator with 5% CO₂.
2. Cell passage is done when the monolayer reaches a confluence of 70% to 80%.
3. Gently rinse the monolayer with 1X PBS to remove waste and debris.
4. Perform trypsin digestion with 1 mL of 0.050% Trypsin for 3 minutes at 37°C.
5. Terminate digestion with 2 mL 1X PBS and transfer the cell suspension into a 15 mL centrifuge tube.
6. Centrifuged at 150 g for 3 minutes.
7. Discard the supernatant gently.
8. Add 1 mL fresh DMEM with 10% FBS into the tube and gently resuspend the cells.
9. To a new 100 mm culture dish, subculture the cell by adopting a ratio of 1:3 to 1:5 in 10 mL 10% FBS DMEM.

Development of acquired gemcitabine Resistance

1. Subculture the cell lines as described in the previous section, except that Gemcitabine is added into the culture medium after deducing the IC₅₀ from mock cells.
2. Gemcitabine was used in a stepwise increase manner to 3 µM for the development of resistance.
3. Incubate the cells at 37°C and 5% CO₂, cell passage is performed upon a confluence of 70 to 80%.
4. The resistance level is assessed periodically by MTT cytotoxic assay.

MTT cytotoxicity Assay

Cell seeding of mock and Gemcitabine resistant (GemR) PANC-1 cells

• Gently rinse the monolayer with 1X PBS to remove waste and debris.
• Perform trypsin digestion with 1 mL of 0.050% Trypsin for 3 minutes at 37°C.
• Terminate digestion with 2 mL 1X PBS and transfer the cell suspension into a 15 mL centrifuge tube for cell washing.
• Centrifuge at 150 g for 3 minutes then discard the supernatant gently.
• Prepare a cell suspension by adding 1 mL of 10% FBS DMEM into the washed cell platelet.
• Acquire 2 μL of cell suspension and add into 18 μL trypan blue solution, calculate and record the dilution factor.
• Perform cell counting after trypan blue staining then deduce the cell concentration:

  average # of viable cells =

  viable cells

  squares numbers (4)

  Dilution Factor =

  volume of cells added

  total volume of cell

  Cell concentration (no. of cell/mL) = average number of counted viable cells × Dilution factor × 10⁴

• Prepare a cell dilution of 2.5 × 104 cells/mL in 10% FBS DMEM.
• To each well of the 96-well plate, dispense 200 μL of cell suspension. The final cell concentration is 5000 cells/well.
• Incubate the cell-seeded plate for 24 hours at 37 °C and 5% CO₂, remove the medium after 24 hours for the addition of Gemcitabine solution.

Addition of Gemcitabine

1. Prepare serial dilutions of Gemcitabine with the use of 2.5% FBS DMEM medium according to the need.
2. The concentration range of Gemcitabine employed is between 0 and 1 mM.
3. Schematic representation of Gemcitabine usage on a 96-well plate:



After the addition of Gemcitabine, incubate the plate for 72 hours at 37 °C, 5% CO₂, then perform MTT assay to check the cell viability.

Detection of cell viability by MTT

1. After the 72 hours incubation, gently remove the medium with the Gemcitabine from each well.
2. Apply 110 μL of 5 mg/mL MTT solution to each well.
3. Incubate the 96 Well Plates for 3 hours at 37 °C, 5% CO₂.
4. Gently remove the MTT solution from each well.
5. Add 100 μL DMSO to each well and agitate the plate for 10 minutes on a rotary shaker.
6. Check for complete solubilization of the purple formazan crystals and measure the absorbance at 570 nm.
7. Data obtained is processed and analysed with the use of GraphPad Prism (Version 10).

1. HER2 Surface Staining
   • Cell Lines: PANC-1 (Mock, Gemcitabine-resistant)
   • Antibody: IgG-APC
   • Dilution: 1:200 for PANC-1
   • Incubation: 30 minutes at 4°C for PANC-1
2. EGFR Surface Staining
   • Fixation/Permeabilization: BD Cytofix/Perm for 30 minutes at 4°C
   • Cell Lines: PANC-1 (Mock, Gemcitabine-resistant)
   • Antibody: CST EGFR
   • Dilution: 1:200
   • Incubation: 30 minutes at 4°C
   • Additional Staining: AZ488 Rabbit (1:1000) for 30 minutes, followed by AF488 Rabbit (1:500) for 20 minutes at 4°C
3. Surface Staining
   • Cell Lines: PANC-1 (Parental, Resistant)
   • Antibody: CST EGFR
   • Dilution: 1:200
   • Incubation: 30 minutes at 4°C
   • Additional Staining: AF488 Rabbit (1:500) for 30 minutes at 4°C

Staining Procedure

1. 2 x 105 cells were stained by APC-conjugated HER2 (#324408, Biolegend) antibodies in dilution of 1:500 in PBS with 2% FBS at 4 °C for 15 minutes.
2. 2 x 105 cells were first fixed and permeabilized using BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (#554714, BD Biosciences). The fixed cells were washed and incubated with EGFR primary antibodies (#4267, Cell Signaling Technology) in dilution of 1:200 for 30 minutes at 4 °C.
3. Then incubated with anti-Rabbit IgG secondary antibody conjugated with Alexa FluorTM488 (#A-11008, ThermoFisher Scientific) in dilution of 1:500 minutes for 20 minutes at 4 °C. Isotype-matched immunoglobulins served as controls.
4. Samples were analyzed using BD Accuri C6 flow cytometer and FACSDiva software (BD Biosciences).

b) Bliss Analysis

1. Calculate the individual and combination drug effects on cell viability based on the assay results. This may involve comparing the drug-treated groups to the control group and determining the percentage of cell viability.
2. Apply the Bliss independence model to analyze the combination drug effects. This model predicts the expected combined effect based on the assumption of non-interaction between the drugs. The Bliss score can be calculated using the formula:
   Bliss score = (A + B) - (A x B), where A and B represent the effects of the individual drugs.
3. Compare the observed combination effects with the predicted Bliss score. Positive deviations from the Bliss score indicate synergy, while negative deviations indicate antagonism.