Day 1:
1. Coat glass plates with 100 µL collagen (1:50 in PBS) for 3 hr in 37 ⁰C tissue culture incubator.
2. Remove collagen, plate 4,500-6,000 RPE-1 cells in RPE-1 growth media in each well.
Late afternoon of Day 2:
1. Prepare transfection mixtures, Opti-MEM/Plasmid and Opti-MEM/Lipofectamine, incubating separately for 5 min at room temperature. Then mix together and incubate for 30 min at room temperature.
Opti-MEM/Plasmid Mixture (volumes per well of 96 well plate):
9 µL Opti-MEM
1 µL of 1:1 CDK plasmid:CDK binding protein plasmid (0.5 µg of each plasmid)
OR
Opti-MEM/Plasmid Mixture (volumes per well of 96 well plate):
9 µL Opti-MEM
1 µL of 1:1:1 CDK plasmid:cyclin plasmid:CIP/KIP plasmid (0.5 µg of CDK plasmid, 0.25 µg of cyclin and CIP/KIP plasmid)
Opti-MEM/Lipofectamine Mixture (volumes per well of 96 well plate):
9.75 µL Opti-MEM
0.25 µL Lipofectamine 2000
2. Add 80 µL of RPE-1 growth media per well of transfection mixture prepared above.
3. Remove growth media from cells prepared the previous day, and add 100 µL of the transfection mixture per well.
4. Incubate cells with transfection mixture for 2-3 hr in 37 ⁰C tissue culture incubator.
5. Replace transfection mixture with 100 µL of RPE-1 growth media per well.
Day 3:
1. Change media, adding 100 µL fresh RPE-1 media per well.
2. Prepare drug cocktails to prevent cell movement.
Drug Cocktail I:
247 µL RPE-1 Growth Media
3 µL 10 mM Y27631
Drug Cocktail II:
237 µL RPE-1 Growth Media
1 µL 10 mM Y27631
8 µL 1 mM Jasplakinolide
2 µL 5 mM Latrunculin B
2 µL 500 µg/mL nocodazole
3. Add 50 µL Drug Cocktail I to one well of 96 well you plan to image. Incubate 20 min at 37 ⁰C.
4. Add 50 µL Drug Cocktail II to the same well of 96 well plate. Incubate 30 min at 37 ⁰C.
5. While incubating with Drug Cocktail II, identify cells to image in step 6.
5a. Using widefield/Yokogawa spinning-disc confocal microscope and 60x water immersion objective described in the Equipment section, find the plane of the cell in which localization of the nuclear periphery can be observed.
5b. Use stage controls to find cells meeting the following criteria. The x,y positions of cells were saved for imaging in Step 6.
i. The cell expresses all of the constructs of interest.
ii. Sufficient expression of the CDK- Δ50 lamin A fluorescent construct that a ring of localization can be observed for the interacting protein of interest.
iii. Note that the nuclei with Δ50 lamin A expressed can form irregular contours and multi-lobed nuclei. Mild cases of these phenotypes can be used for this method.
6. Image and photobleach cells identified in step 5 for no more than 2 hr after adding drug Cocktail II.
6a. Cells were imaged every second for the first 35 seconds, followed by every 5 seconds for the next 90 seconds, and every 30 seconds until the end of the imaging. A total of 15 minutes was imaged for most conditions.
6b. Cells were photobleached after the first five seconds of imaging. Photobleaching regions were drawn as an ellipse with widths, and heights of 1 µm.
Analysis
1. In Slidebook 6.13 software, using the draw regions tools, draw the following three regions:
i. the region of interest (ROI), where the photobleaching at the nuclear lamin occurred.
ii. the reference region (REF), a distant nucleoplasmic region with the cell.
iii. the background (BG), a region absent of any cells.
2. Export the data for these three regions across the imaging time course as an excel document.
3. Generate a corrected curve for the fluorescent signal of interest using the following equation:
CORRECTED CURVE= (ROI-BG)/(REF-BG) '
4. Normalize the corrected curve from 0 to 1 by subtracting the first post-photobleaching value from all time points, then dividing all time points by the average of the pre-photobleaching corrected values.
5. For a given condition, the normalized corrected curves of at least 10 cells imaged on at least two separate days of experimental setup was averaged and the standard deviation plotted.