A polarized cell system amenable to subcellular resolution imaging of influenza virus infection

Prepare the repellent layer and seed the Caco-2/TC7 cells

Dissolve the Gelrite solution (0.8% Gelrite + 0.1% MgSO₄ x7H₂O w/v in PBS) in the microwave oven.

Distribute the Gelrite solution (2.5mL/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 0h 10m 0s.

Wash a 100 mm diameter culture dish of subconfluent Caco-2/TC7 cells with 5mL of PBS.

Add 1mL of trypsin and incubate 0h 5m 0s at 37°C.

Add 9mL 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.

Count the cells. Prepare 2mL of a cell suspension at 2,25 x 10⁵ cells/mL.

Add 50µL 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.

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.

After three days, filter out the non-attached cells

Place a 70 µm cell strainer over a 50 mL tube.

Collect the beads suspension and pass it through the cell strainer. Discard the flow-through.

Flip the filter with sterile tweezers over the Gelrite layer, add 2mL of fresh D10 medium on the top of the flipped filter to recover the beads.

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.

Transfer the Cytodex beads from the Gelrite layer to a 15mL tube. Let them sediment at the bottom of the tube.

Remove gently the medium, add 1mL of prewarmed (37°C) DMEM for washing, and let the beads sediment at the bottom of the tube.

Repeat the washing step once, then transfer the beads to a 1.5mL tube.

Let the beads sediment and remove gently the medium, resuspend the beads in 250µL 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.

Incubate the beads at 37°C for 1h 0m 0s, and gently tap the tube with fingertip every 15 mn to resuspend the beads.

Let the beads sediment at the bottom of the tube, remove the viral inoculum, add 1mL of PBS for washing and let the beads sediment at the bottom of the tube.

Remove the PBS, add 1mL 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).

Fix the cells

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 0h 20m 0s.

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.

Repeat the washing step twice, then transfer the beads to a 1.5mL tube.

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.

Wash the beads 2x with 1mL PBS as described above.

Immunostain the cells

Let the beads sediment at the bottom of the tube. Remove the PBS and add 500µL of the blocking buffer. Place the tube on a spinning wheel at 4°C for 1h 0m 0s, with a rotation speed of 15rpm,0h 0m 0s.

Place an adhesive secure-seal spacer on a clean glass microscope slide.

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.

Cover the drop with a sterile #1.5 round glass microscope coverslip. Protect from light and let at Room temperature 2h 0m 0s and seal with nail polish.

Let the beads sediment at the bottom of the tube. Remove the blocking buffer and add 250µL of the primary antibody diluted in the immuno-staining buffer. Place the tube on a spinning wheel at 4°C 1h 0m 0s, with a rotation speed of 15rpm,0h 0m 0s.

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

Let the beads sediment at the bottom of the tube. Remove the PBS-0.05% Tween 20 and add 250µL of the secondary antibody diluted in the immunostaining buffer. Place the tube on a spinning wheel at 4°C for 2h 0m 0s, with a rotation speed of 15rpm,0h 0m 0s.

Wash the beads 2x with 1mL of PBS- 0.05% Tween 20 as described above.

Wash the beads 2x with 1mL of PBS as described above.

Wash the beads 2x with 1mL of distilled water as described above.

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 50µL of Vectashield mounting medium over the beads pellet, avoiding the formation of bubbles.

Gently tap the tube with fingertip to resuspend the beads.

Acquire confocal images.

Select the 40x NA 1,3 oil immersion objective and apply appropriate oil.

Install sample on the slide holder of the microscope.

Create the lightpath configuration corresponding to the staining used. Create three imaging sequence:

1. DAPI : Excitation 405 nm, Emission : 416-501 nm
2. PALS-1 : Excitation 598 nm, Emission : 608-643 nm
3. NP : Excitation 646 nm, Emission : 656-705 nm

Find your sample, adjust focus.

Laser power and gain settings will be set according to the intensity of the labelling in order not to saturate the signal.

Define your Z-stack by selecting the top and the bottom of your sample. Define Z step size to 0,35 µm.

Set acquisition parameters: Set frame size to 1024x1024 pixels (0,3 µm/pixel), scan speed to 400 Hz in a bi-directional mode.

Start image acquisition.

Acquire confocal images at high magnification.

Select the HC PL APO 93x NA 1,3 glycerol immersion objective and apply appropriate glycerol.

Install sample on the slide holder of the microscope.

Adapt the motorized objective colar to match the sample index, using reflection mode in XZY mode.

Create the lightpath configuration corresponding to the staining used. Create three imaging sequence:

1. Atto 647N : Excitation 670 nm, Emission : 654-719 nm
2. AlexaFluor 594 : Excitation 594 nm, Emission : 604-641 nm
3. Stargreen : Excitation 470 nm, Emission : 505-561 nm

Find your sample, adjust focus.

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.

Define your Z-stack by selecting the top and the bottom of your sample. Define Z step size to 0,18 µm.

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).

Fix the cells.

Remove the medium carefully while leaving the beads at the bottom of the well. Resuspend the beads in 250µL of PBS-3% PFA-0.1% glutaraldehyde. Leave at 4°C 2h 0m 0s.

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 250µL of PHEM-2.5% glutaraldehyde. Leave at 4°C for 24h 0m 0s.

Let the beads sediment at the bottom of the tube. Remove gently the PHEM-2.5% glutaraldehyde, add 1mL of PHEM, incubate for 0h 5m 0s at Room temperature.

Repeat the PHEM washing 2x.

Perform uranyl acetate staining.

Let the beads sediment at the bottom of the tube. Remove gently the PHEM, add 250µL of the PHEM-1% Osmium tetroxide-1.5% potassium ferrocyanide post-fixation buffer. Incubate for 1h 0m 0s at Room temperature.

Wash 3x in PHEM as described above.

Wash 3x in filtered distilled water, with 5 mn incubation times as described above.

Let the beads sediment at the bottom of the tube. Remove gently the water, add 250µL of water supplemented with 0.2% tannic acid. Incubate 0h 30m 0s at Room temperature.

Wash 3x in filtered distilled water, with 0h 5m 0s incubation times as described above.

Let the beads sediment at the bottom of the tube. Remove gently the water, add 250µL of water supplemented with 2% osmium. Incubate 0h 30m 0s at Room temperature.

Wash 3x in filtered distilled water, with 0h 5m 0s incubation times as described above.

Let the beads sediment at the bottom of the tube. Remove gently the water, add 250µL of 25% ethanol in water (v/v)- 1% Uranyl Acetate. Incubate 1h 0m 0s at Room temperature (protect from light).

Embed the samples.

Let the beads sediment at the bottom of the tube. Remove gently the uranyl acetate solution. Add 1mL of 50% Ethanol in water (v/v). Incubate 0h 5m 0s at Room temperature.

Repeat step 16.8 with 75% ethanol in water (v/v).

Repeat step 16.8 with 100 % ethanol.

Prepare the Epoxy resin at Room temperature.(https://www.emsdiasum.com/docs/technical/datasheet/14120)..)

Let the beads sediment at the bottom of the tube and remove gently the ethanol with a micropipette.

Add 200µL of Epoxy resin onto the beads and homogenize slowly with back-and-forth movements with the micropipette.

Let the beads sediment at the bottom of the tube. Remove 150µL of Epoxy resin and add 150µL of fresh Epoxy resin.

Wait 3h 0m 0s of impregnation time with the tubes open under the chemical hood.

Incubate 3h 0m 0s at Room temperature to allow polymerization of the resin.

Section the samples with an ultramicrotome.

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.

Cut semi-thin sections (1 µm thick). The sections float on the water in the bowl of the HISTO diamond knife.

Collect the sections in a drop of water with a handle and place them on a glass slide.

Stain the semi-thin sections by adding a drop of 50µL of toluidine blue (5% p/v) on the sections and incubating the samples 2 mn on a hot plate (60°C).

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.

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.

Cut ultrathin sections (70 nm thick). The sections float on the water in the bowl of the Ultra 45° diamond knife.

Carefully collect 2 sections on a carboned grids.

Place the grids with thin sections into the transmission electron microscope sample box.

Place the shaped block into the chuck on the ultramicrotome and tighten it firmly.

Place the HISTO diamond sectioning knife on the sectioning knife holder stage on the ultramicrotome.

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.

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.

Adjust the knife angle so that the two parallel sides of the trapezoid sample are parallel to the blade.

Move the knife to align the available part with the block surface.

Raise the block until it stops slightly above the knife blade.

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.

Staining of ultrathin sections.

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.

For each grid, add one drop (about 100µL) of water supplementmented with 4% Uranyl Acetate, filtered (0.22µm), on the parafilm.

Place gently the grid (section side) on the droplet surface and let it stand for 0h 40m 0s. Protect it from light.

For each grid, add three drops (about 200µL) 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 0h 0m 10s before transferring a grid to the next droplet.

For each grid, add one drop of water supplemented with 1% lead citrate, filtered (0.22 µm), on a clean area of the parafilm.

Place the grid on the top of the droplet and let it stand for 0h 5m 0s.

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 0h 0m 20s before transferring a grid to the next droplet.

After drying the grids on a filter paper, place them in the transmission electron microscope sample box.

Electron microscopy imaging.

Acquire images with a transmission electron microscope at 120 kV.

Collect images of cells on beads.