Protocol 1: Pulse-chase experiments for long-term imaging with Nikon Ti2-E

Materials and Reagents

1. 10-cm Petri dish (Corning, catalog number: 430167)
2. Micropipette tips, serological pipettes, pipette aids, and microtubes for liquid handling
   
   1. TipOne^® 1,000 µl XL (catalog number: S1122-1830)
   2. TipOne^® 200 µl (catalog number: S1120-8810)
   3. TipOne^® 20 µl filter tips (catalog number: S1120-1810) or SARSTEDT pipette tip 10 µl (catalog number: 70.1130.600)
   4. Fisherbrand^TM 5 ml serological pipets (catalog number: 13-676-10H)
   5. Fisherbrand^TM 10 ml serological pipets (catalog number: 13-676-10J)
   6. Fisherbrand^TM 25 ml serological pipets (catalog number: 13-676-10K)
   7. Drummond Pipet-Aid XL (catalog number: 4-000-205)
   8. Microtube 1.5 ml (SARSTEDT, catalog number: 72.690.001)
3. Nunc 35-mm glass bottom (#1.5 or 0.16-0.19 mm thickness) dishes with 27 mm viewing area (Thermo Fisher Scientific, catalog number: 150682)
   Notes:
   
   1. Only open packages inside a biosafety cabinet and reseal any remaining dishes to avoid contamination.
   2. Use any dish or slide format that is compatible with an inverted microscope. Match the thickness of the glass bottom with the suggested thickness found on the microscope’s objective (typically #1.5 or 0.16-0.19 mm thickness). We prefer the dishes listed above because of their large viewing area. The cell lines used in this protocol can grow on glass substrates, but surface treatment (e.g., Poly-Lysine coating) may be necessary for other cell lines.
4. HuH-7 cell line (Japanese Collection of Research Bioresources [JCBR], catalog number: JCBR 0403)
   Note: HuH-7 is a well-differentiated hepatocyte-derived cellular carcinoma cell line. It was originally taken from a liver tumor in a Japanese male in 1982 (Nakabayashi et al., 1982). The cells used in the creation of this protocol were obtained from John McLauchlan (Centre for Virus Research, Glasgow).
5. A plasmid that encodes SNAP_f^®, a SNAP-tag protein (NEB, catalog number: N9183S; or as part of the SNAP-Cell^® Starter Kit, NEB, catalog number: E9100S)
6. For initial validation: plasmid that encodes pSNAP_f-Cox8A control plasmid (also part of the SNAP-Cell^® Starter Kit, NEB, catalog number: E9100S)
7. Trypsin EDTA solution (Sigma, catalog number: T3924-500ML)
8. Specific reagents for our cell line (for basic tissue culture), which we derived from the hepatoma cell line HuH-7
   
   1. Dulbecco's modified Eagle's medium (Sigma, catalog number: D6429-500ML)
   2. 100% Fetal Calf Serum (Gibco, catalog number: 10500-064)
   3. Gibco MEM Nonessential amino acids solution (100x), store at 4 °C up to 24 months from the date of manufacture (Thermo Fisher Scientific, catalog number: 11140050) 
   4. Gibco HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) buffer (100x), store at 4 °C up to 24 months from the date of manufacture (Thermo Fisher Scientific, catalog number: 15630130) 
9. FluoroBrite DMEM, store at 4 °C up to 24 months from the date of manufacture (Thermo Fisher Scientific, catalog number: A1896701)
10. Cell-permeable SNAP-Cell^® 647-SiR (New England Biolabs, catalog number: S9102S)
    Notes: 
    
    1. Package contains 30 nmol of the substrate. Resuspend with 50 µl of sterile dimethyl sulfoxide (DMSO, Fisher Bioreagents^TM, catalog number: BP231-100) to make up a stock solution, which can be stored at -20 °C. We have used stock solutions that have been stored for up to 3 years. However, the manufacturer’s recommended shelf-life is three months dissolved in DMSO and two years dry.
    2. NEB also offers cell-impermeable SNAP probes (‘Cell Surface’ Probes); use these probes for studies of viral proteins that accumulate at the surface of host cells. 
11. Optional: Red fluorescent or orange fluorescent cellular dye (e.g., MitoTracker^TM Orange CMTMRos, Thermo Fisher Scientific, catalog number: M7510)
    Note: To prepare a stock solution, dissolve lyophilized MitoTracker^TM probe in DMSO to a final concentration of 1 mM and store frozen and protected from light.
12. Molecular Probes Invitrogen ProLong Live Antifade Reagent, for live cell imaging (Thermo Fisher Scientific, catalog number: P36975)
    Notes:
    
    1. Store at 2-8 °C for short-term storage and ≤ -20 °C for long-term storage.
    2. According to the manufacturer’s instructions, use the product within 30 days when stored at 2-8 °C. When stored at ≤ -20 °C, the product is stable for at least six months with up to four freeze-thaw cycles.
13. Glutamax-I (Thermo Fisher Scientific, catalog number: 35050061)
14. Live cell imaging solution (see Recipes)
15. Tissue culture medium containing serum (see Recipes)
    

Equipment

1. Air-displacement micropipettes
   
   1. Starlab ErgoOne^® 100-1,000 µl Single-Channel Pipette (catalog number: S7110-1000) or Gilson F123602 PIPETMAN Classic Pipet P1000 (Fisher Scientific, catalog number: 10387322)
   2. Starlab ErgoOne^® 20-200 µl Single-Channel Pipette (catalog number: S7100-2200) or Gilson F123615 PIPETMAN Classic Pipet P100 (Fisher Scientific, catalog number: 10442412)
   3. Starlab ErgoOne^® 2-20 µl Single-Channel Pipette (catalog number: S7100-0220) or Starlab ErgoOne^® 0.1-2.5 µl Single-Channel Pipette (catalog number: S7100-0125) or Gilson F144801 PIPETMAN Classic Pipet P2 (Fisher Scientific, catalog number: 10635313) or Gilson F123600 PIPETMAN Classic Pipet P20 (Fisher Scientific, catalog number: 10082012)
2. Equipment for basic cell culture techniques and aseptic procedures, i.e.,
   1. Biosafety cabinet (e.g., Thermo Scientific Holten Safe 2010 Model 1.2, catalog number: 8207071100)
   2. Humidified incubator set to 37 °C (Panasonic Incusafe, catalog number: MCO-20AIC)
   Note: Any manufacturer and model of a biosafety cabinet will do, but the chosen biosafety cabinet needs to be appropriate for the containment of cells and viruses. When handling infectious viruses, use the proper facilities, practices, and procedures. Refer to local and international guidelines for laboratory biosafety. 
3. -20 °C freezer (Labcold, catalog number: RLCF1520)
4. Water bath (e.g., Clifton, catalog number: NE2-8D; however, any water bath set to 37 °C may be used)
5. Table-top microcentrifuge, 4 °C to room temperature (RT), max speed ≥ 12,000 x g (Eppendorf, catalog number: 5424R)
   Note: For spinning down precipitate that may form during extended storage of SNAP probes.
6. Live-cell imaging microscope for long-term imaging. For example, a Nikon Ti2-E inverted widefield microscope (for a similar setup, contact your local Nikon representative to create a customized order for the microscope system)
   
   1. Nikon Ti2-E inverted microscope stand
   2. Motorized stage (standard Ti2 encoded motorized XY stage)
   3. Lumencor Spectra X LED light source
   4. CFI Plan Apo Lambda 60x oil/1.4 NA objective
   5. Photometric Prime 95B sCMOS monochrome camera
   6. Semrock 32 mm filter (Green) for Nikon Ti2-E: GFP-4050B Filter cube with Ex 466/40 single-band bandpass filter (product code: FF01-466/40-25), DM495 dichroic beamsplitter (product code: FF495-Di03-25x36), BA525/50 single-band bandpass filter (product code: FF03-525/50-25)
   7. Semrock 32 mm filter (Red) for Nikon Ti2-E: Cy3-4040C Filter cube with Ex 531/40 single-band bandpass filter (product code: FF01-531/40-25), Dichroic Mirror DM 562 (product code: FF562-Di03-25x36, Barrier Filter: BA 593/40 (product code: FF01-593/40-25)
   8. Semrock 32 mm filter (Far-Red) for Nikon Ti2-E: Cy5-4040C Filter cube with Ex 628/40 single-band bandpass filter (product ID: FF02-628/40-25), Single Band Emitter (product ID: FF01-692/40-25), Single Band Dichroic (product ID: FF660-Di02-25x36) 
   9. Okolab stage top incubator, catalog number: H301-NIKON-NZ100/200/500-N. Set to 37 °C with 5% CO₂ (any manufacturer and model that can be mounted on a Nikon Ti2 motorized stage will do)
   10. Perfect Focus System (PFS)
   Notes:
   
   1. The above list is not exhaustive but rather lists the essential components that have been useful in the creation of Protocol 1. Consult with your local Nikon representative for the remaining components of a customized microscope system.
   2. We prefer to image cells in open tissue-culture dishes with a coverslip-like glass bottom, which necessitates an inverted microscope stand. 
   3. As Protocol 1 is a powerful approach for long-term imaging of the labeled protein, it is important to keep cells in focus and correct for focus drift caused by thermal and mechanical conditions. This live-cell imaging system included Nikon’s fourth-generation Perfect Focus System (PFS), which monitors the partial reflection of a low-power infrared laser beam on the interface between the dish’s glass bottom and liquid media above. This feature provides continuous and real-time focus drift correction. 
   4. A large (25 mm x 25 mm) field of view (FOV) enables increased data throughput and capture of additional cells that would be outside the normal FOV. 
   5. We preferred an LED light source over laser-based illumination. LED illumination limited photobleaching and phototoxicity; ‘gentle’ imaging was essential during acquisition of five-dimensional datasets (5-D: multi-color 3-D Z-stacks over time). 
   6. A motorized microscope stage and the ability to record multiple positions during one experiment further increased the throughput of the imaging system.

Software

1. Nikon NIS Elements AR imaging software
   Nikon NIS Elements AR imaging software controls all components of the microscope. Use NIS Elements to set-up parameters for acquisition of 5-D datasets. Users can also create rendered videos within NIS Elements and save individual frames of 5-D data. For our widefield images, we also used a Richardson-Lucy algorithm (set to 10 iterations) to deconvolve datasets within the Elements software.
   

Procedure