Sample Preparation for In Situ Cryotomography of Mammalian Cells

The scope of this research is to develop a methodology for sample preparation that is both accessible and reliably reproducible. By achieving this, the power of direct cellular cryo-electron tomography can be used to answer a wide variety of biological questions. The challenges of grid preparation are extensive.

Cells do not readily attach to grids, and manipulation can result in damage to the carbon layer, resulting in the loss of imageable grid areas. Additionally, the prevalence of thin cellular protrusions is reduced without attention to proper sample preparation conditions. Current protocols typically require specialized equipment to solve the major bottlenecks of seeding, such as micro-patterning and 3D-printed holders for cell attachment and manipulation, respectively.

This protocol provides solutions with greater accessibility while still maintaining flexibility for these specialized tools to be used for downstream applications. To begin preparing the electron microscopy grids for cellular cryo tomography, place the grid loaded with perforated carbon support film in a glow discharge device with the carbon side of the grid facing up. Then glow discharge the grid at 15 milliamperes under 0.39 millibar atmospheric pressure for 45 seconds.

Once done, place the grid in a clean container lined with filter paper. Next, transfer the grid, along with fibronectin, phosphate-buffered saline or PBS, plates, and tweezers, to a biosafety cabinet. Using a micropipette, cast two generously sized dots of bovine fibronectin onto a sterile surface, like a six-well plate lid.

Ensure the dots are large enough to envelop the entire grid. Then obtain 5/15 tweezers, and treat both sides of the grid with a fibronectin adherence solution. Once treated with bovine fibronectin, the grid will become sticky.

Gently touch the non-carbon surface of the grid to the bottom of an empty well in a six-well plate, and release the tweezers to attach the grid to the new surface. Next, incubate the grid for 30 minutes in the fibronectin adherence solution To prevent the grids from drying, add one to two drops of adherence solution on top of the grids using a micropipette every 15 minutes. Following the incubation, remove the excess adherent solution with a micropipette.

Wash the grids by adding drops of PBS directly on top of the grids using the micropipette two to three times. Sterilize the grids under UV light in a biosafety cabinet for one hour. Position the grids as close to the UV as possible for maximum sterilization.

Prevent the grids from drying during sterilization by micropipetting one to two drops of PBS on top of the grid every 10 minutes. To begin cell seeding, count the cells after detaching them using mechanical or enzymatic methods. Using a micropipette, add the calculated number of cells per well in the six-well plate containing pre-prepared electron microscopy grids while avoiding bubbles.

Ensure to maintain 1.5 to 2.0 milliliters of cell suspension per well. For the transfection of HIV molecular clones, incubate the cells for 16 to 24 hours at 37 degrees Celsius. Add 50 microliters of serum-free DMEM and one microgram of DNA to a clean microcentrifuge tube.

For each well, dilute three microliters of transfection reagent in serum-free DMEM into a separate, clean microcentrifuge tube. Homogenize the mixtures separately using a micropipette. Then add the transfection reagent mixture to the DNA mixture via micropipette, and incubate for 15 minutes at room temperature.

After that, add 100 microliters of the transfection reagent and DNA mixture dropwise into each well directly on top of the grids. Replace the growth medium 16 to 24 hours after transfection. 24 hours following the co-transfection, phase light microscopy and fluorescent light microscopy showed that all the grids had minimal tearing in the carbon layer.

Cells on both the mock grids and the co-transfected grids contained viable cells in multiple grid squares. Before plunge-freezing the electron microscopy grids containing the cells, inspect the grids under an inverted optical microscope. Record the cell densities on each grid to adjust blotting times during plunge-freezing.

Warm two to three milliliters of phosphate-buffered saline or PBS in an empty plate at 37 degrees Celsius. For gold fiducials preparation, pipette 50 microliters of 10 nanometer colloidal gold bead solution into a clean 1.5-milliliter tube. Add two microliters of bovine serum albumin to it, and homogenize by micropipetting repeatedly.

Centrifuge the tube at 15, 000 to 20, 000 g for 15 minutes. Carefully remove the supernatant without disturbing the pellet using a micropipette. Resuspend the pellet in 50 microliters of PBS, and centrifuge the tube again, as previously demonstrated.

Aspirate four microliters of the pellet using a 10-microliter tip, and transfer it to a clean 1.5-milliliter tube. Set up the environmental chamber at 95%humidity and 30 degrees Celsius. Using 5/15 tweezers, select a grid and wash it with PBS.

Transfer the washed grid to plunging tweezers. Once the grid is secured, remove the 5/15 tweezers, and slide the clamp on the plunging tweezers. Insert the grid into the plunge freezer while keeping the clamp secure.

Add one microliter of gold fiducials to the backside of the grid. Then add two to three microliters of PBS to the carbon side of the grid. Utilize automated blotting to blot the backside of the grid before plunge-freezing into liquid ethane.

A cryo-correlative light and electron microscopy image of transfected U-2 OS cells revealed the presence of HIV-producing cells. An electron cryo tomography image showed multiple HIV particles budding from the plasma membrane of U-2 OS cells.