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NOTE: The ovarian cancer cell line, OVCAR3, was used in these steps, but this protocol is broadly applicable to multiple other cell lines, including those derived from non-ovarian sources. A schematic of the protocol is shown in Figure 1.
1. Plating the cells
- Make poly-L-lysine coated coverslips.
- Add autoclaved 12 mm diameter coverslips to a 50 mL conical tube with poly-L-lysine solution, and place on a rocker for 15 min.
- Aspirate the solution in a tissue culture hood. Wash the coverslips by adding sterile water and place the tube containing the coverslips back on the rocker for 5 min. Repeat this wash step three times.
- Spread the coated coverslips on a sterile dish and aspirate any remaining water. Let the coverslips dry in the tissue culture hood for 1 h or until no water droplets remain. Once dry, seal the dish with parafilm, and place at 4 °C.
- Place one poly-L-lysine-coated coverslip in each well of a 24-well plate. The use of poly-lysine coverslips is critical to prevent the detachment of the cells from the coverslips during the pre-extraction step.
- Trypsinize OVCAR3 cells once they reach 70%-80% confluency.
- To trypsinize a 10 cm culture dish that is 70%-80% confluent, aspirate the medium from the plate, and wash with 5-7 mL of phosphate-buffered saline (PBS).
- Aspirate the PBS, add 1 mL of 0.25% trypsin, and place the dish in a 37 °C incubator for 8-10 min, or until cells lift off from the bottom of the plate.
- Collect the cells with 5-10 mL of medium and add to a conical tube.
- Count the cells manually with a hemocytometer using standard procedures.
- Make a dilution of 25,000 cells/mL and add 1 mL of the cells on poly-L-lysine coverslips placed in the wells of 24-well plates. For any other cell line, determine a number such that the cells are about 70%-80% confluent after three population doublings.
- Grow the cells in the culture medium under standard conditions.
2. Pulsing cells with IdU
- For the cells to properly spread onto the coverslips, one population doubling is enough. After one population doubling, pulse the cells with 10 µM 5-iodo-2'-deoxyuridine (IdU) (see Table of Materials on how to reconstitute IdU) for the two subsequent population doublings.
NOTE: We have tried different thymidine analogs, including bromodeoxyuridine (BrdU), 5-chloro-2′-deoxyuridine (CIdU), and IdU. Amongst the three analogs, IdU gives the best signal-to-noise ratio. Comparative images of cells pulsed with the three different analogs are shown in Figure 2. We recommend the use of two negative controls: (i) a no IdU pulsed sample, and (ii) a no primary antibody control. If the formation of ssDNA needs to be assessed due to a given treatment, we recommend adding the drug after the first round of IdU doubling.
- After pulsing with IdU for two population doublings, harvest the cells for imaging.
- Replace the medium with ice-cold 0.5% PBSTx (PBS + 0.5% Triton X-100) on ice for 5 min. This pre-extraction step helps to release cytoplasmic and non-chromatin-bound proteins, leaving chromatin-bound proteins intact. Certain cell lines can easily peel off during pre-extraction. In such a scenario one can use 0.5% CSK buffer (10 mM PIPES (pH 6.8),100 mM sodium chloride (NaCl), 300 mM sucrose, 3 mM magnesium chloride (MgCl2), 1 mM ethylene glycol tetraacetic acid (EGTA) and 0.5% Triton X-100).
3. Fixation
- Aspirate PBSTx and incubate for 15 min with 3% paraformaldehyde (PFA) (Table of Materials) at room temperature, followed by three to four washes with 1x PBS. The fixed cells can be kept at 4 °C until further steps.
CAUTION: PFA is highly toxic and carcinogenic. Avoid contact with skin, eyes, and mucous membranes. Perform all the steps with PFA in a fume hood, and dispose of the materials properly.
4. Permeabilization and blocking
- After fixation, permeabilize the cells using 0.5% PBSTx on ice for 5 min. Use enough volume to cover the entire coverslip (typically between 500 µL and 1 mL).
- Wash the cells three to four times with 0.2% PBST (1X PBS + 0.2% Tween-20) at room temperature. One mL of PBST is enough for washing each well. Perform the washes back-to-back without any incubations.
- Aspirate the PBST and block the samples using 5% bovine serum albumin, BSA (Table of Materials) made in 1x PBS (blocking buffer) for 30 min at room temperature.
5. Immunostaining with the IdU antibody
- For immunostaining, prepare a humidified chamber (wet paper towel on a flat-bottom Tupperware). Cover the lid of the 24-well plate with parafilm, place in the humidified chamber, and lay down the coverslips on the plate lid.
- Prepare the anti-BrdU primary mouse antibody (Table of Materials) by diluting it 1:200 in the blocking buffer from step 4.2. The anti-BrdU antibody has previously been shown to detect IdU.
- Add 60 µL of IdU antibody dilution on the top of the coverslips. Incubate the coverslips for 1 h at 37 °C.
- Alternatively, use less antibody solution if the coverslips are flipped onto a drop (20-25 µL) of 1:200 antibody dilution pipetted onto parafilm. This also decreases the likelihood of the solution drying out during the incubation.
- After the 1 h incubation, aspirate the primary antibody. Return the coverslips back to a 24-well plate, and wash them four times with 0.2% PBST.
- For the secondary antibody, use the same humidified chamber described in step 5.1. Dilute anti-mouse conjugated secondary antibody (Table of Materials) in the blocking buffer (1:200). Add 60 µL of secondary antibody to the coverslips, and incubate at room temperature in the dark for 1 h.This secondary antibody is light-sensitive.
- Aspirate the secondary antibody. Return the coverslips back to the 24-well plate, and wash four times with 0.2% PBST.
- Label microscope slides, and mount the coverslips on the slides with 4',6-diamidino-2-phenylindole (DAPI) mounting medium (Table of Materials). Store slides in the dark at room temperature for 24 h. The 24 h incubation is recommended if the mounting medium needs to be cured or hardened.
NOTE: The slides can then be stored in 4 °C before being imaged on a fluorescence microscope. A representative image is shown in Figure 3.
