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The goal of this summer project is to observe binding between the RBD of SARS-CoV-2 Spike Protein and human ACE2 using fluorescence microscopy.
Supplementary Information
Cell line used: B16-F10
Complete Growth Medium: RPMI 1640 + 10% FBS + Pen-Strep (50 I.U./mL - 50 ug/mL)
FBS: Gibco (Fisher Scientific Catalog No, A5670401)
Pen-Strep: Gibco (Fisher Scientific Catalog No, 15-070-063)
Transfection Protocol is based on ThermoFisher Lipofectamine™ 3000 Transfection Reagent.
✸ DNA amount is increased in these experiments for ensuring enough amount of protein expression.
B16-F10 Passaging
Materials
70% Ethanol
1 mL, 100 uL micropipette and pipette tips
Liquid waste container
Pre-warming container (60 mm and 35 mm cell culture dish -or- 14 mL falcon tube)
Complete Growth Medium at 37°C: RPMI 1640 + 10% FBS
0.25% Trypsin - EDTA at RT (room temperature)
Growing B16-F10 cell
Procedure
Add complete growth medium 5 mL and 3 mL to the new 60 mm and 30 mm culture dish, respectively. Then, pre-warm the medium at 37°C for at least 15 min.
Remove and discard old culture medium.
Briefly rinse the cell layer with Trypsin solution 1 mL.
Remove and discard the solution.
Add Trypsin solution 1 mL to the dish.
Incubate the cells at 37°C for 5 min and observe the cells under microscope to check the cell layer is dispersed.
✸Note: To avoid clumping do not agitate the cells by hitting or shaking the dish while waiting for the cells to detach.
Add the prepared pre-warmed complete growth medium 3 mL and homogenize the cells by gently pipetting.
Spread the cell suspension 40 uL to the new 60 mm dish containing a new complete growth medium.
Mix the solution well to make a homogeneous cell solution and check under a microscope to ensure the cell amount and distribution is sufficient.
✸Note: Do Not move the dish in a circular motion or the cells will collect in the middle of the dish and will not be evenly spread out.
Mark the culture dish with the date, the procedure, and your initials, and incubate at 37°C.
Calu-3 Passaging
Materials
70% Ethanol
1 mL, 100 uL micropipette and pipette tips
Liquid waste container
Pre-warming container (60 mm and 35 mm cell culture dish -or- 14 mL falcon tube)
Complete Growth Medium at 37°C: EMEM + 10% FBS
0.25% Trypsin - EDTA at RT (room temperature)
Growing Calu-3 cell
Procedure
Add complete growth medium 5 mL and 3 mL to the new 60 mm and 30 mm culture dish, respectively. Then, pre-warm the medium at 37°C for at least 15 min.
Remove and discard old culture medium.
Briefly rinse the cell layer with Trypsin solution 1 mL.
Remove and discard the solution.
Add Trypsin solution 1 mL to the dish.
Incubate the cells at 37°C for 10 min and observe the cells under microscope to check the cell layer is dispersed.
✸Note: To avoid clumping do not agitate the cells by hitting or shaking the dish while waiting for the cells to detach.
Add complete growth medium 3 mL and homogenize the cells by gently pipetting.
Spread the cell suspension 400 uL to the new 60 mm dish containing a new complete growth medium.
Mix the solution well to make a homogeneous cell solution and check under a microscope to ensure the cell amount and distribution is sufficient.
✸Note: Do Not move the dish in a circular motion or the cells will collect in the middle of the dish and will not be evenly spread out.
Mark the culture dish and incubate at 37°C.
B16-F10 Cell Growth Medium Replacement
✸ Growth medium needs to be replaced with new medium twice a week (3 days after passaging -> 2 days after the 1st replacement).
Prewarm a new complete growth medium 5 mL
Discard the old medium in a culture dish
Add the prewarmed medium into the dish
Incubate at 37°C.
Cell Pre-seeding for Transfection (24-well plate)
Materials
100 uL, 10 uL micropipette and pipette tips
Hemocytometer
24-well plate
Complete Growth medium
Cell suspension from cell passaging step: After adding growth medium to Trypsinized cell
forceps (autoclaved)
glass (autoclaved)
Procedure
Add autoclaved glass in each well (forceps used must also be autoclaved)
Add complete growth medium 400 uL into each well of 24-well plate.
Take 10 uL of cell suspension and inject into one side of hemocytometer
Note: Cell suspension must be homogenous right before you pipette it. Mix it well by pipetting the solution up and down multiple times.
Place the hemocytometer on a microscope, count the number of cells, and calculate concentration of the cell suspension.
Note: # of cell / mL = average # of cells in one square X 10^4 / mL
Calculate the volume of cell suspension that is needed to seed 1.0 X 10^5 cells per well
Mix the cell suspension well by pipetting the solution up and down multiple times.
Add the calculated volume of the cell solution into each well of the 24-well plate.
Mix the solution in each well by pipetting up and down.
Incubate the 24-well plate at 37°C overnight.
Cell Pre-seeding for Transfection (96-well plate)
Materials
100 uL, 10 uL micropipette and pipette tips
Hemocytometer
96-well plate
Complete Growth medium
Cell suspension from cell passaging step: After adding growth medium to Trypsinized cell
Procedure
Add complete growth medium 100 uL into each well of 96-well plate.
Take 10 uL of ~90% confluence cell suspension and inject into one side of hemocytometer
Note: Cell suspension must be homogenous right before you pipette it. Mix it well by pipetting the solution up and down multiple times.
Place the hemocytometer on a microscope, count the number of cells, and calculate concentration of the cell suspension.
Note: # of cell / mL = average # of cells in one square X 10^4 / mL
Calculate the volume of cell suspension that is needed to seed 2.0 X 10^4 cells per well
Mix the cell suspension well by pipetting the solution up and down multiple times.
Add the calculated volume of the cell solution into each well of the 96-well plate.
Mix the solution in each well by pipetting up and down.
Incubate the 96-well plate at 37°C overnight.
I. B16-F10 cell transfection with eGFP
✸ This procedure is for transfection in 24-well plate.
Seed 0.5 x 10^5 cells and add complete growth media (500 uL).
Incubate cells overnight at 37°C and 5% CO2.
Dilute Lipofectamine™ 3000 Reagent (0.75 uL or 1.5 uL) in Opti-MEM™ Medium (25 uL) – Mix well
Dilute eGFP pcDNA (0.5 ~ 2.0 ug) in Opti-MEM™ Medium (25 uL), then add P3000™ Reagent (2 µL/µg DNA) – Mix well
✸ For the negative control, DNA and P3000 is not added to the Opti-MEM medium.
Add Diluted DNA to the tube of Diluted Lipofectamine™ 3000 Reagent (1:1 ratio)
Incubate for 10–15 minutes at room temperature.
Add DNA-lipid complex to cells
Incubate cells for 1–3 days at 37°C and 5% CO2. Then, analyze transfected cells.
Day 2, DNA: 0.5 ug, Lipofectamine: 0.75 uL
(400uL of growth media substituted with 100uL of Opti-MEM at Day 1)
Day 2, DNA: 1.0 ug, Lipofectamine: 1.5 uL
Day 2, DNA: 2.0 ug, Lipofectamine: 1.5 uL
Day 2, Negative Control
II. B16-F10 cell transfection with human ACE2
✸ This procedure is for transfection in 96-well plate.
Seed 1~4 x 10^4 cells and add complete growth media (100 uL).
Incubate cells overnight at 37°C and 5% CO2.
Dilute Lipofectamine™ 3000 Reagent (0.3 uL) in Opti-MEM™ Medium (5 uL) – Mix well
Dilute human ACE2 pcDNA (0.8 ~ 1.2 ug) in Opti-MEM™ Medium (5 uL), then add P3000™ Reagent (2 µL/µg DNA) – Mix well
Add Diluted DNA to the tube of Diluted Lipofectamine™ 3000 Reagent (1:1 ratio)
Incubate for 10–15 minutes at room temperature.
Add DNA-lipid complex to cells
Incubate cells for 2–3 days at 37°C and 5% CO2. Then, analyze transfected cells.
III. Human ACE2 antibody binding : Fixation included (Updated for using PFA)
Remove the cell culture medium and wash the cells 3 times using 1X PBS (100 uL per wash).
Fix cells using 4% Paraformaldehyde (100 uL) for 10 min at RT.
Remove Paraformaldehyde and wash the cells 3 times using 1X PBS (100 uL per wash).
Add 5% BSA in RPMI1640 (100 uL) and incubate the cells at room temperature for 60 min. (Alternatively, the cells can be incubated overnight in 5% BSA before proceeding to immunostaining.)
Remove the BSA solution.
Add the desired concentration of human ACE2 antibody diluted in 1% BSA - RPMI1640 (40 uL) to the cells and incubate for 3 hours at room temperature on a 3D rotator while being protected from light. (1:100 and 1:50 dilution used in this experiment.)
Remove antibody solution and wash the cells 3 times using 1X PBS (100 uL per wash).
Add 1X PBS (50 uL) to prevent cells from being dried.
III. Human ACE2 antibody binding : Fixation included (24-well plate)
Remove the cell culture medium and wash the cells 3 times using 1X PBS (200 uL per wash).
Fix cells using 4% Paraformaldehye (200 uL) for 10 min at RT.
Remove methanol and wash the cells 3 times using 1X PBS (100 uL per wash).
Add 5% BSA in 1X PBS (200 uL) and incubate the cells at room temperature for 60 min. (Alternatively, the cells can be incubated overnight in 5% BSA before proceeding to immunostaining.)
Remove the BSA solution.
Add the desired concentration of human ACE2 antibody diluted in 1% BSA - 1X PBS solution (100 uL) to the cells and incubate for 3 hours at room temperature on a 3D rotator while being protected from light. (1:100 and 1:50 dilution used in this experiment.)
Remove antibody solution and wash the cells 3 times using 1X PBS (200 uL per wash).
Add 1X PBS (200 uL) to prevent cells from being dried.
B16-F10 Antibody Incubation (24-well plate)
B16-F10 Antibody Incubation (24-well plate)
III. Human ACE2 antibody binding : Fixation included (96-well plate)
Remove the cell culture medium and wash the cells 3 times using 1X PBS (100 uL per wash).
Fix cells using 100% ice-cold methanol (100 uL) for 5 min at –20°C.
Remove methanol and wash the cells 3 times using 1X PBS (100 uL per wash).
Add 5% BSA in 1X PBS (100 uL) and incubate the cells at room temperature for 60 min. (Alternatively, the cells can be incubated overnight in 5% BSA before proceeding to immunostaining.)
Remove the BSA solution.
Add the desired concentration of human ACE2 antibody diluted in 1% BSA - 1X PBS solution (40 uL) to the cells and incubate for 3 hours at room temperature on a 3D rotator while being protected from light. (1:100 and 1:50 dilution used in this experiment.)
Remove antibody solution and wash the cells 3 times using 1X PBS (100 uL per wash).
Add 1X PBS (50 uL) to prevent cells from being dried.
Day 2, DNA: 1.2 ug, Antibody 1:100
Day 2, DNA: 1.2 ug, Antibody 1:50
Day 2, DNA: 1.2 ug, Antibody 1:50, magnification X40
Day 2, Negative Control
IV. Human ACE2 antibody binding : Non-Fixed
Remove the cell culture medium and wash the cells 3 times using 1X PBS (100 uL per wash).
Add the desired concentration of human ACE2 antibody diluted in 1X PBS solution (40 uL) to the cells and incubate for 3 hours at room temperature on a 3D rotator while being protected from light. (1:100, 1:50, 1:25 dilution used in this experiment.)
Remove antibody solution and wash the cells 3 times using 1X PBS (100 uL per wash).
Add 1X PBS (50 uL) to prevent cells from being dried.
V. SARS-CoV-2 RBD binding : Fixation included (24-well plate)
Remove the cell culture medium and wash the cells 3 times using 1X PBS (200 uL per wash).
Add the desired concentration of SARS-CoV-2 RBD diluted in 1X PBS solution (100 uL) to the cells and incubate for 1.5 hours at room temperature while being protected from light. (100nM, 500nM used in this experiment.)
Remove RBD solution and wash the cells 3 times using 1X PBS (200 uL per wash).
Fix cells using 4% Paraformaldehye (200 uL) for 10 min at RT.
Remove Paraformaldehyde and wash the cells 3 times using 1X PBS (200 uL per wash). Paraformaldehyde should be discarded into designated Paraformaldehyde disposal container.
Add 1X PBS (200 uL) to prevent cells from being dried.
V. SARS-CoV-2 RBD binding : Fixation included (96-well plate)
Remove the cell culture medium and wash the cells 3 times using 1X PBS (100 uL per wash).
Add the desired concentration of SARS-CoV-2 RBD diluted in 1X PBS solution (40 uL) to the cells and incubate for 1.5 hours at room temperature while being protected from light. (2.5 ug/mL, 5.0 ug/mL, 10.0 ug/mL used in this experiment.)
Remove RBD solution and wash the cells 3 times using 1X PBS (100 uL per wash).
Fix cells using 100% ice-cold methanol (100 uL) for 5 min at –20°C.
Remove methanol and wash the cells 3 times using 1X PBS (100 uL per wash).
Add 1X PBS (50 uL) to prevent cells from being dried.
VI. SARS-CoV-2 RBD binding : Non-Fixed
Remove the cell culture medium and wash the cells 3 times using 1X PBS (100 uL per wash).
Add the desired concentration of SARS-CoV-2 RBD diluted in 1X PBS solution (40 uL) to the cells and incubate for 1.5 hours at room temperature while being protected from light. (2.5 ug/mL, 5.0 ug/mL, 10.0 ug/mL used in this experiment.)
Remove RBD solution and wash the cells 3 times using 1X PBS (100 uL per wash).
Add 1X PBS (50 uL) to prevent cells from being dried.
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