Time-Lapse Into Immunofluorescence Imaging Using a Gridded Dish

Materials and reagents

Biological materials

1. Mouse embryonic fibroblast wild-type NLS-GFP (MEF WT NLS-GFP) described in previous works [15,18,19]

Reagents

1. Dulbecco’s modified Eagle’s medium (DMEM) [+] 4.5 g/L glucose, L-glutamine, sodium pyruvate (Corning, catalog number: 45000-304)

2. Phosphate-buffered saline (PBS) 1× [-] calcium, magnesium (Corning, catalog number: 45000-446)

3. Characterized fetal bovine serum (FBS) (U.S.) (Cytiva HyClone^TM, catalog number: SH3007103)

4. Penicillin-streptomycin (pen/strep) solution (Corning, catalog number: MT30002CI)

5. 16% paraformaldehyde (PFA) aqueous solution, EM grade (Electron Microscopy Sciences, catalog number: 50-980-487)

6. Bovine serum albumin (BSA) fraction V (Fisher BioReagents, catalog number: BP1605-100)

7. Triton X-100 (VWR Life Science, catalog number: 9002-93-01)

8. Tween 20 molecular biology grade (Promega, catalog number: H5152)

9. 0.25% Trypsin, 0.1% EDTA in HBSS, no calcium, magnesium, and sodium bicarbonate (Corning, catalog number: MT25053CI)

10. Primary and secondary antibodies of choice

11. Hoechst (Invitrogen, catalog number: H3570)

Solutions

1. DMEM medium complete (see Recipes)

2. 4% PFA solution (see Recipes)

3. Triton X-100 solution (see Recipes)

4. Tween 20 solution (see Recipes)

5. BSA solution (see Recipes)

Recipes

1. DMEM medium complete

Thaw the frozen FBS and pen/strep in a 37 °C bead bath. Add 5.5 mL of pen/strep and 50 mL of FBS to 500 mL of DMEM. Mix by inverting. Store at 4 °C.

2. 4% PFA solution

Add 3 mL of 16% PFA to 9 mL of PBS in a conical tube. Ensure the conical tube is covered in aluminum foil to avoid light exposure. Mix by inverting. Store at room temperature for up to one month.

Note: Handle PFA with gloves in a fume hood and dispose of PFA in a hazardous waste container.

3. Triton X-100 solution

Add 400 μL of Triton X-100 to 400 mL of PBS. Mix using a stir bar. Store at room temperature for up to one month.

4. Tween 20 solution

Add 240 μL of Tween 20 to 400 mL of PBS. Mix using a stir bar. Store at room temperature for up to one month.

5. BSA solution

Add 500 mg of BSA to 25 mL of PBS. Mix by inverting. The BSA solution can be stored at 4 °C for up to one week.

Laboratory supplies

1. 60 mm surface-treated tissue culture dishes (Fisher, catalog number: FB012921)

2. 35 mm glass bottom dish with 14 mm micro-well #1.5 gridded cover glass (Cellvis, catalog number: D35-14-1.5GI)

3. 15 mL conical tubes (VWR International, catalog number: 470225-000)

4. 50 mL conical tubes (VWR International, catalog number: 470225-004)

5. 2 mL serological pipettes (Fisher, catalog number: 13-678-11C)

6. 5 mL serological pipettes (Fisher, catalog number: 13-678-11D)

7. 10 mL serological pipettes (Fisher, catalog number: 13-678-11E)

8. 2 μL pipette tips (Gilson, catalog number: 76178-284)

9. 20 μL pipette tips (Gilson, catalog number: 76178-282)

10. 1,000 μL pipette tips (Gilson, catalog number: 76180-360)

11. Parafilm (Amcor, catalog number: 13-374-5)

Equipment

1. Incubator (Heracell VIOS 160i Tri-Gas CO₂ Incubator) (Thermo Scientific, catalog number: 51033720)

2. Pipet-Aid XP2 (Drummond Scientific Company, catalog number: 4-000-501)

3. Nutating mixer (VWR International, catalog number: 82007-202)

4. Light microscope [Nikon Instruments Ti-2E microscope, Orca Fusion Gen III Camera, Lumencor Aura III light engine, Perfect Focus System, TMC CleanBench air table, with 40× air objective (N.A 0.75, W.D. 0.66, MRH00401) or use with Crest V3 Spinning Disk Confocal]

5. Stage heater (Okolab Stage Heater, model: H401-T-Controller)

6. Stage top incubator (Okolab Stage Top Incubator, model: H301)

7. Refrigerator (4 °C)

Procedure

A. Preparing the gridded dish

1. Plate MEF WT NLS-GFP in 37 °C DMEM medium complete (see Recipe 1) on a single-well gridded dish. Incubate overnight at 37 °C with 5.0% CO₂ to allow cells to adhere.

Note: Plate cells to reach 50%–70% confluency on the day of time-lapse imaging. Take into account drug incubation times.

B. Time-lapse imaging

1. Allow the microscope stage to reach 37 °C and 5.0% CO₂.

2. Transfer the gridded dish from the incubator to the microscope.

Note: Orient the dish so that the grid is right side up. Check the dish under transmitted light to ensure the dish is oriented correctly (Figure 1A). With a marker, mark the bottom of the dish so the orientation can be matched during immunofluorescence imaging.

Figure 1. Gridded dish preparation for time-lapse imaging. (A) Representative image of a gridded dish in a coordinate-oriented right side up under transmitted light (scale bar = 50 μm). (B) Representative image of a field of view of MEF WT NLS-GFP cells in a coordinate under fluorescein isothiocyanate (FITC) light (excitation 498 nm, emission 517 nm) (scale bar = 10 μm).

3. Select fields of view with 70% confluency (Figure 1B). Note the coordinate for each field of view, e.g., A7, CS, etc.

Note: Select adjacent coordinates to make cell matching easier. Ensure that the fields of view have no overlapping cells.

4. Run the time-lapse according to standard protocol.

Critical: Perform the following steps as quickly as possible after the time-lapse ends to minimize cell movement.

5. Remove cells from the microscope and transfer them back to the cell culture hood. Replace DMEM with 3 mL of PBS.

6. To fix cells, prepare 4% PFA (see Recipe 2) in a 15 mL conical tube in the fume hood. From this point forward, keep the dish covered with foil to prevent light exposure.

7. Remove PBS and add 3 mL of 4% PFA to the gridded dish. Dispose of PFA in a hazardous waste container. Cover in foil and let sit for 15 min at room temperature.

Caution: Wash gently to minimize cell movement.

8. Remove 4% PFA and wash with 3 mL of PBS for 5 min. Repeat two times.

Pause point: Fixed cells can be stored in 3 mL of PBS at 4 °C for up to one month before the protocol is resumed. Wrap cells in parafilm to prevent evaporation. Incomplete fixation of cells may result in shorter storage times.

C. Immunofluorescence

1. Remove PBS and add 3 mL of Triton X (see Recipe 3). Let it sit for 15 min at room temperature.

2. Remove Triton X and add 3 mL of Tween 20 (see Recipe 4). Let it sit for 5 min at room temperature.

3. Remove Tween 20 and wash with 3 mL of PBS for 5 min. Repeat two times.

4. To block cells, prepare BSA (see Recipe 5) in a 50 mL conical tube. Allow to dissolve completely at 4 °C.

5. Remove PBS from the dish and add 3 mL of BSA. Let it sit for 1 h at room temperature.

6. Remove BSA and add 1,000 μL of primary antibody solution according to the standard protocol. Wrap in parafilm and let it sit overnight at 4 °C.

Note: Pipette the antibody solution directly onto the cells in the center portion of the coverslip, not into the outer plastic ring.

7. Remove the primary antibody solution and wash with 3 mL of PBS for 5 min. Repeat two times.

8. Remove PBS and add 1,000 μL of secondary antibody solution according to the standard protocol. Let it sit on a nutating mixer for 1 h at room temperature.

Note: Pipette the antibody solution directly onto the cells in the center portion of the coverslip, not into the outer plastic ring.

9. Remove the secondary antibody solution and wash with 3 mL of PBS for 5 min. Repeat two times.

10. Remove PBS and add 1,000 μL of Hoechst DNA stain solution according to the standard protocol. Let it sit at room temperature for 15 min.

Note: Pipette the staining solution directly onto the cells in the center portion of the coverslip, not into the outer plastic ring.

11. Remove the DNA stain solution and wash with 3 mL of PBS for 5 min. Repeat two times.

12. Replace PBS with 3 mL of fresh PBS.

D. Locating cells via immunofluorescence imaging

1. Place the dish onto the microscope in the same orientation as during time-lapse imaging. Use the previously drawn mark as a guide.

2. Open time-lapse images in the desired software and select the last frame of each field of view to use as a visual reference.

3. Locate the recorded coordinates from fields of view during the time-lapse in transmitted light and adjust so that the cells match the last frame in the time-lapse (Figure 2).

4. Locate all of the matching fields of view from the time-lapse. Image matching fields of view according to the standard protocol. Name each immunofluorescence field of view to match its time-lapse counterpart.

Figure 2. Matching time-lapse field of view during immunofluorescence imaging. (A) Representative image of the last frame of a time-lapse field of view under fluorescein isothiocyanate (FITC) light (excitation 498 nm, emission 517 nm). Matching immunofluorescence images stained with Hoechst, lamin B1, and merged. Scale bars, 10 μm. (B) Top row: example images from time-lapse imaging of a blebbed unruptured nucleus and subsequent immunofluorescence (NLS-GFP images from time 0, 1, 2, and 3 h; white arrow denotes bleb). Bottom row: example images from time-lapse imaging of a blebbed ruptured nucleus and subsequent immunofluorescence (NLS-GFP images from time 0, 2, 4, and 16 min; white arrow denotes bleb). Scale bars, 10 μm.

Validation of protocol

This protocol has been used and validated in the following research articles:

• Chu et al. [1]. Lamin B loss in nuclear blebs is rupture dependent while increased DNA damage is rupture independent. Journal of Cell Science (Figure 1A).

• Bunner et al. [2]. Decreased DNA density is a better indicator of a nuclear bleb than lamin B loss. Journal of Cell Science.

General notes and troubleshooting

General notes

1. Depending on the length of the time-lapse, this protocol likely takes two days. To complete the protocol in one day, primary antibodies can be incubated at 37 °C for 2 h or according to the standard protocol. The rest of the protocol can then be completed as normal, following three washes with PBS.

Troubleshooting

Problem 1: Cells have either moved or disappeared from their original position during immunofluorescence imaging.

Possible causes: Waiting too long to fix after the time-lapse ends; harsh washing.

Solutions: Fix the cells as soon as the time-lapse ends. Gently wash the cells by pipetting liquids in and out of the outer plastic ring rather than directly onto the cells in the center.

Acknowledgments

Conceptualization, A.S., N.L., C.G.C. Investigation, N.L., C.G.C. Writing—Original Draft, N.L., C.G.C. Writing—Review & Editing, A.S., N.L., C.G.C. Funding acquisition, A.S. Supervision, A.S. This work was primarily supported by NIH NIGMS grant Maximizing Investigators’ Research Award R35GM154928. This protocol was first validated in Chu et al. [1]. We would like to thank Kelsey Prince for streamlining the first iteration of time-lapse into immunofluorescence using a gridded dish. We would also like to thank Maddy Clark and BioRender for creating the images used in the Graphical overview.

Competing interests

The authors declare no competing interests.

References

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