TITLE: A polarized cell system amenable to subcellular resolution imaging  of influenza virus infection
AUTHORS: Brault Jean-Baptiste, Thouvenot Catherine, Cannata Serio Magda, Paisant Sylvain, Fernandes Julien, Gény David, Danglot Lydia, Mallet Adeline, Naffakh Nadia

[DESCRIPTION]
This protocol details a polarized cell system amenable to subcellular resolution imaging of influenza virus infection.

[STEPS]
SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
1. Prepare the repellent layer and seed the Caco-2/TC7 cells
SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
1.1. Dissolve the Gelrite solution (0.8% Gelrite + 0.1% MgSO4 x7H2O w/v in PBS) in the microwave oven.
SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
1.2. Distribute the Gelrite solution (2.5 mL/well) in a 35 mm dish or a well of a 6-well plate. Let it solidify and cool down at Room temperature for about 10 min.

SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
1.3. Wash a 100 mm diameter culture dish of subconfluent Caco-2/TC7 cells with 5 mL of PBS.

SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
1.4. Add 1 mL of trypsin and incubate 5 min at 37 °C.

SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
1.5. Add 9 mL of DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin (D10 medium). Pipet up and down to homogenize the cell suspension.

SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
1.6. Count the cells. Prepare 2 mL of a cell suspension at 2,25 x 105 cells/mL.

SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
1.7. Add 50 µL of the Cytodex 3 suspension (~1500 beads/50 µL) to the cell suspension, mix gently and add to the Gelrite layer. Place into the cell incubator onto a rubber pad to attenuate the incubator’s vibrations.

SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
2. The next day, homogenize the beads suspension by gently pipetting up and down 5x with a 1000 µL pipetman tip, place them back in the cell incubator.
SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
3. After three days, filter out the non-attached cells
SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
3.1. Place a 70 µm cell strainer over a 50 mL tube.
SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
3.2. Collect the beads suspension and pass it through the cell strainer. Discard the flow-through.
SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
3.3. Flip the filter with sterile tweezers over the Gelrite layer, add 2 mL of fresh D10 medium on the top of the flipped filter to recover the beads.

SECTION: SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
3.4. Change the medium twice a week by removing the medium carefully while leaving the beads at the surface of the Gelrite layer, and replacing it with fresh D10 medium. Keep going for 10-15 days.
SECTION: INFLUENZA A VIRUS INFECTION (in a BSL2+ laboratory)
4. Transfer the Cytodex beads from the Gelrite layer to a 15mL tube. Let them sediment at the bottom of the tube.
SECTION: INFLUENZA A VIRUS INFECTION (in a BSL2+ laboratory)
5. Remove gently the medium, add 1 mL of prewarmed (37 °C) DMEM for washing, and let the beads sediment at the bottom of the tube.

SECTION: INFLUENZA A VIRUS INFECTION (in a BSL2+ laboratory)
6. Repeat the washing step once, then transfer the beads to a 1.5mL tube.
SECTION: INFLUENZA A VIRUS INFECTION (in a BSL2+ laboratory)
7. Let the beads sediment and remove gently the medium, resuspend the beads in 250 µL of a suspension of Influenza A virus (A/WSN/1933(H1N1)) at a high titer. Given the estimated numbers of ~ 1500 beads/sample and ~400 cells/bead, a  ~2,5 x107 PFU/mL titre will lead to an  MOI of ~10 PFU/cell. 

SECTION: INFLUENZA A VIRUS INFECTION (in a BSL2+ laboratory)
8. Incubate the beads at 37 °C for 60 min, and gently tap the tube with fingertip every 15 mn to resuspend the beads.

SECTION: INFLUENZA A VIRUS INFECTION (in a BSL2+ laboratory)
9. Let the beads sediment at the bottom of the tube, remove the viral inoculum, add 1 mL of PBS for washing and let the beads sediment at the bottom of the tube.

SECTION: INFLUENZA A VIRUS INFECTION (in a BSL2+ laboratory)
10. Remove the PBS, add 1 mL of DMEM supplemented with 2% FBS, resuspend the beads and place them in a well of a 12-well culture plate. Place the plate in a cell incubator at 37 °C for the selected period of time (typically, 2-6 hours to investigate early stages of the viral life cycle, 8-24 hours to investigate late stages).

SECTION: CONFOCAL IMAGING
11. Fix the cells
SECTION: CONFOCAL IMAGING
11.1. Remove the medium carefully while leaving the beads at the bottom of the well. Resuspend the beads in 250µL of PBS-4% PFA. Incubate at room temperature for 20 min.

SECTION: CONFOCAL IMAGING
11.2. Transfer the beads suspension in a 1.5mL tube. Let the beads sediment at the bottom of the tube. Remove gently the PBS-4% PFA, add 1mL of PBS for washing, and let the beads sediment at the bottom of the tube.
SECTION: CONFOCAL IMAGING
11.3. Repeat the washing step twice, then transfer the beads to a 1.5mL tube. 
 
 
SECTION: CONFOCAL IMAGING
11.4. Let the beads sediment at the bottom of the tube. Remove the PBS and add 1mL of PBS-50mM NH4Cl for 10 minutes at room temperature, to quench the residual PFA.
SECTION: CONFOCAL IMAGING
11.5. Wash the beads 2x with 1mL PBS as described above.
SECTION: CONFOCAL IMAGING
12. Immunostain the cells
SECTION: CONFOCAL IMAGING
12.1. Let the beads sediment at the bottom of the tube. Remove the PBS and add 500 µL of the blocking buffer. Place the tube on a spinning wheel at 4 °C for 60 min, with a rotation speed of 15 rpm.

SECTION: CONFOCAL IMAGING
12.2. Let the beads sediment at the bottom of the tube. Remove the blocking buffer and add 250 µL of the primary antibody diluted in the immuno-staining buffer. Place the tube on a spinning wheel at 4 °C 60 min, with a rotation speed of 15 rpm. 

SECTION: CONFOCAL IMAGING
12.3. The next day, wash the beads 3x with 1 mL of PBS-0.05% Tween 20 as described above.

SECTION: CONFOCAL IMAGING
12.4. Let the beads sediment at the bottom of the tube. Remove the PBS-0.05% Tween 20 and add 250 µL of the secondary antibody diluted in the immunostaining buffer. Place the tube on a spinning wheel at 4 °C for 120 min, with a rotation speed of 15 rpm.

SECTION: CONFOCAL IMAGING
12.5. Wash the beads 2x with 1 mL of PBS- 0.05% Tween 20 as described above.

SECTION: CONFOCAL IMAGING
12.6. Wash the beads 2x with 1 mL of PBS as described above.

SECTION: CONFOCAL IMAGING
12.7. Wash the beads 2x with 1 mL of distilled water as described above.

SECTION: CONFOCAL IMAGING
12.8. Let the beads sediment at the bottom of the tube and remove the water. Use a very thin 10µL pipetman tip to remove residual water and to dry out the beads pellet. Immediately add 50 µL of Vectashield mounting medium over the beads pellet, avoiding the formation of bubbles.

SECTION: CONFOCAL IMAGING
12.9. Gently tap the tube with fingertip to resuspend the beads.
SECTION: CONFOCAL IMAGING
12.10. Place an adhesive secure-seal spacer on a clean glass microscope slide.
SECTION: CONFOCAL IMAGING
12.11. Cut the extremity of 200µL pipetman tip to widen it, gently pipet out the beads resuspended in Vectashield mounting medium, avoiding the formation of bubbles. Drop them at the center of the spacer on the glass microscope slide.

SECTION: CONFOCAL IMAGING
12.12. Cover the drop with a sterile #1.5 round glass microscope coverslip. Protect from light and let at Room temperature 120 min and seal with nail polish.

SECTION: CONFOCAL IMAGING
13. Acquire confocal images.
SECTION: CONFOCAL IMAGING
13.1. Select the 40x NA 1,3 oil immersion objective and apply appropriate oil.
SECTION: CONFOCAL IMAGING
13.2. Install sample on the slide holder of the microscope.
SECTION: CONFOCAL IMAGING
13.3. Create the lightpath configuration corresponding to the staining used. Create three imaging sequence:
DAPI : Excitation 405 nm, Emission : 416-501 nm
PALS-1 : Excitation 598 nm, Emission : 608-643 nm
NP : Excitation 646 nm, Emission : 656-705 nm
SECTION: CONFOCAL IMAGING
13.4. Find your sample, adjust focus.
SECTION: CONFOCAL IMAGING
13.5. Laser power and gain settings will be set according to the intensity of the labelling in order not to saturate the signal.
SECTION: CONFOCAL IMAGING
13.6. Define your Z-stack by selecting the top and the bottom of your sample. Define Z step size to 0,35 µm.
SECTION: CONFOCAL IMAGING
13.7. Set acquisition parameters: Set frame size to 1024x1024 pixels (0,3 µm/pixel), scan speed to 400 Hz in a bi-directional mode.
SECTION: CONFOCAL IMAGING
13.8. Start image acquisition.
SECTION: CONFOCAL IMAGING
14. Acquire confocal images at high magnification.
SECTION: CONFOCAL IMAGING
14.1. Select the HC PL APO 93x NA 1,3 glycerol immersion objective and apply appropriate  glycerol.
SECTION: CONFOCAL IMAGING
14.2. Install sample on the slide holder of the microscope.
SECTION: CONFOCAL IMAGING
14.3. Adapt the motorized objective colar to match the sample index, using reflection mode in XZY mode.
SECTION: CONFOCAL IMAGING
14.4. Create the lightpath configuration corresponding to the staining used. Create three imaging sequence:
Atto 647N : Excitation 670 nm, Emission : 654-719 nm
AlexaFluor 594 : Excitation 594 nm, Emission : 604-641 nm
Stargreen : Excitation 470 nm, Emission : 505-561 nm
SECTION: CONFOCAL IMAGING
14.5. Find your sample, adjust focus.
SECTION: CONFOCAL IMAGING
14.6. Laser power and gain settings will be set according to the intensity of the labelling in order not to saturate the signal. HyD detectors are used to favor sensitivity.
SECTION: CONFOCAL IMAGING
14.7. Define your Z-stack by selecting the top and the bottom of your sample. Define Z step size to 0,18 µm.
SECTION: CONFOCAL IMAGING
14.8. Set acquisition parameters: scan speed to 400 Hz in a bi-directional mode, Frame Average2. Adapt zoom, matrix size and Z step to be in ideal sampling (here pixel size 85 nm).
SECTION: ELECTRON MICROSCOPY IMAGING
15. Fix the cells.
SECTION: ELECTRON MICROSCOPY IMAGING
15.1. Remove the medium carefully while leaving the beads at the bottom of the well. Resuspend the beads in 250 µL of PBS-3% PFA-0.1% glutaraldehyde. Leave at 4 °C 120 min.

SECTION: ELECTRON MICROSCOPY IMAGING
15.2. Transfer the beads suspension in a 1.5mL tube. Let the beads sediment at the bottom of the tube. Remove gently the PBS-3% PFA-0.1% glutaraldehyde, add 250 µL of PHEM-2.5% glutaraldehyde. Leave at 4 °C for 1440 min.

 



SECTION: ELECTRON MICROSCOPY IMAGING
15.3. Let the beads sediment at the bottom of the tube. Remove gently the PHEM-2.5% glutaraldehyde, add 1 mL of PHEM, incubate for 5 min at Room temperature.
SECTION: ELECTRON MICROSCOPY IMAGING
15.4. Repeat the PHEM washing 2x.
SECTION: ELECTRON MICROSCOPY IMAGING
16. Perform uranyl acetate staining.
SECTION: ELECTRON MICROSCOPY IMAGING
16.1. Let the beads sediment at the bottom of the tube. Remove gently the PHEM, add 250 µL of the PHEM-1% Osmium tetroxide-1.5% potassium ferrocyanide post-fixation buffer. Incubate for 60 min at Room temperature.

SECTION: ELECTRON MICROSCOPY IMAGING
16.2. Wash 3x in PHEM as described above.
SECTION: ELECTRON MICROSCOPY IMAGING
16.3. Wash 3x in filtered distilled water, with 5 mn incubation times as described above.
SECTION: ELECTRON MICROSCOPY IMAGING
16.4. Let the beads sediment at the bottom of the tube. Remove gently the water, add 250 µL of water supplemented with 0.2% tannic acid. Incubate 30 min at Room temperature.

SECTION: ELECTRON MICROSCOPY IMAGING
16.5. Wash 3x in filtered distilled water, with 5 min incubation times as described above.

SECTION: ELECTRON MICROSCOPY IMAGING
16.6. Let the beads sediment at the bottom of the tube. Remove gently the water, add 250 µL of water supplemented with 2% osmium. Incubate 30 min at Room temperature.

SECTION: ELECTRON MICROSCOPY IMAGING
16.7. Wash 3x in filtered distilled water, with 5 min incubation times as described above.

SECTION: ELECTRON MICROSCOPY IMAGING
16.8. Let the beads sediment at the bottom of the tube. Remove gently the water, add 250 µL of 25% ethanol in water (v/v)- 1% Uranyl Acetate. Incubate 60 min at Room temperature (protect from light).

SECTION: ELECTRON MICROSCOPY IMAGING
17. Embed the samples.
SECTION: ELECTRON MICROSCOPY IMAGING
17.1. Let the beads sediment at the bottom of the tube. Remove gently the uranyl acetate solution. Add 1 mL of 50% Ethanol in water (v/v). Incubate 5 min at Room temperature.

SECTION: ELECTRON MICROSCOPY IMAGING
17.2. Repeat step 16.8 with 75% ethanol in water (v/v).
SECTION: ELECTRON MICROSCOPY IMAGING
17.3. Repeat step 16.8 with 100 % ethanol.
SECTION: ELECTRON MICROSCOPY IMAGING
17.4. Prepare the Epoxy resin at Room temperature.(https://www.emsdiasum.com/docs/technical/datasheet/14120).

SECTION: ELECTRON MICROSCOPY IMAGING
17.5. Let the beads sediment at the bottom of the tube and remove gently the ethanol with a micropipette.
SECTION: ELECTRON MICROSCOPY IMAGING
17.6. Add 200 µL of Epoxy resin onto the beads and homogenize slowly with back-and-forth movements with the micropipette.

SECTION: ELECTRON MICROSCOPY IMAGING
17.7. Let the beads sediment at the bottom of the tube. Remove 150 µL of Epoxy resin and add 150 µL of fresh Epoxy resin.

SECTION: ELECTRON MICROSCOPY IMAGING
17.8. Wait 180 min of impregnation time with the tubes open under the chemical hood.

SECTION: ELECTRON MICROSCOPY IMAGING
17.9. Incubate 180 min at Room temperature to allow polymerization of the resin. 

SECTION: ELECTRON MICROSCOPY IMAGING
18. Section the samples with an ultramicrotome.
SECTION: ELECTRON MICROSCOPY IMAGING
18.1. Use a fresh blade to trim away excess resin from the block underneath the embedded compound eye under the stereomicroscope. Shape the upper part into an equilateral trapezoid, and shape the lower part into a square base.
SECTION: ELECTRON MICROSCOPY IMAGING
18.2. Place the shaped block into the chuck on the ultramicrotome and tighten it firmly.
SECTION: ELECTRON MICROSCOPY IMAGING
18.3. Place the HISTO diamond sectioning knife on the sectioning knife holder stage on the ultramicrotome.
SECTION: ELECTRON MICROSCOPY IMAGING
18.4. Add a little excess water to the groove of the diamond sectioning knife to make it convex and stop when the full length of the diamond blade is soaked.
SECTION: ELECTRON MICROSCOPY IMAGING
18.5. Gently draw a portion of the water under the microscope with a syringe or Pasteur pipette. Adjust the lightning system so that when the liquid surface becomes concave, a curved surface reflecting light can be seen, which is the correct liquid surface and appears white under the microscope.
 

SECTION: ELECTRON MICROSCOPY IMAGING
18.6. Adjust the knife angle so that the two parallel sides of the trapezoid sample are parallel to the blade.
SECTION: ELECTRON MICROSCOPY IMAGING
18.7. Move the knife to align the available part with the block surface.
SECTION: ELECTRON MICROSCOPY IMAGING
18.8. Raise the block until it stops slightly above the knife blade.
SECTION: ELECTRON MICROSCOPY IMAGING
18.9. Set parameters in the control panel: feed of 1000 nm, speed of 1 mm/s, thickness 1 µm. Start the automated movement of the ultramicrotome specimen arm.
SECTION: ELECTRON MICROSCOPY IMAGING
18.10. Cut semi-thin sections (  thick). The sections float on the water in the bowl of the HISTO diamond knife.

SECTION: ELECTRON MICROSCOPY IMAGING
18.11. Collect the sections in a drop of water with a handle and place them on a glass slide.
SECTION: ELECTRON MICROSCOPY IMAGING
18.12. Stain the semi-thin sections by adding a drop of 50 µL of toluidine blue (5% p/v) on the sections and incubating the samples 2 mn on a hot plate (60 °C).

SECTION: ELECTRON MICROSCOPY IMAGING
18.13. Look at the stained semi-thin sections on the glass slide with a photonic microscope. Repeat the steps 18.4 to 18.9 until an area of interest with beads is observed.
SECTION: ELECTRON MICROSCOPY IMAGING
18.14. Halt sectioning and replace the HISTO diamond knife with an Ultra 45° diamond knife. Fill the knife reservoir with fresh distilled water. Adjust the liquid level and sectioning parameters: feed of 70 nm, speed of 1 mm/s.
SECTION: ELECTRON MICROSCOPY IMAGING
18.15. Cut ultrathin sections (70 nm thick). The sections float on the water in the bowl of the Ultra 45° diamond knife.
SECTION: ELECTRON MICROSCOPY IMAGING
18.16. Carefully collect 2 sections on a carboned grids.
SECTION: ELECTRON MICROSCOPY IMAGING
18.17. Place the grids with thin sections into the transmission electron microscope sample box.
SECTION: ELECTRON MICROSCOPY IMAGING
19. Staining of ultrathin sections.
SECTION: ELECTRON MICROSCOPY IMAGING
19.1. Prepare a parafilm of 15 cm in width and place it on a delimited radioactive area, with the clean surface of the parafilm on the upper side.
SECTION: ELECTRON MICROSCOPY IMAGING
19.2. For each grid, add one drop (about 100 µL) of water supplementmented with 4% Uranyl Acetate, filtered (0.22µm), on the parafilm.

SECTION: ELECTRON MICROSCOPY IMAGING
19.3. Place gently the grid (section side) on the droplet surface and let it stand for 40 min. Protect it from light.

SECTION: ELECTRON MICROSCOPY IMAGING
19.4. For each grid, add three drops (about 200 µL) of distilled water on a clean area of the parafilm. Place the grid on the first of the three droplets before moving it on the second and then on the third droplet. Wait 10 s before transferring a grid to the next droplet.

SECTION: ELECTRON MICROSCOPY IMAGING
19.5. For each grid, add one drop  of water supplemented with 1% lead citrate, filtered (0.22 µm), on a clean area of the parafilm.
SECTION: ELECTRON MICROSCOPY IMAGING
19.6. Place the grid on the top of the droplet and let it stand for 5 min.

SECTION: ELECTRON MICROSCOPY IMAGING
19.7. For each grid, add  five drops of fresh distilled water on a clean area of the parafilm. Wash the grids by placing them successively on the five droplets, as in step 19.4. Wait 20 s before transferring a grid to the next droplet.

SECTION: ELECTRON MICROSCOPY IMAGING
19.8. After drying the grids on a filter paper, place them in the transmission electron microscope sample box.
SECTION: ELECTRON MICROSCOPY IMAGING
20. Electron microscopy imaging.
SECTION: ELECTRON MICROSCOPY IMAGING
20.1. Acquire images with a transmission electron microscope at 120 kV.
SECTION: ELECTRON MICROSCOPY IMAGING
20.2. Collect images of cells on beads.