An Immunofluorescence-Based Method to Quantify Replication Stress in Cancer Cells

Overview

The video demonstrates an immunofluorescence-based approach for quantifying replication stress in cancer cells. Hydroxyurea-treated cells produce single-stranded DNA, detected through immunofluorescence. The count of green fluorescence foci indicates the level of replication stress in cancer cells.

Protocol

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

1. Make poly-L-lysine coated coverslips.
   1. 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.
   2. 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.
   3. 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.
2. 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.
3. Trypsinize OVCAR3 cells once they reach 70%-80% confluency.
   1. 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).
   2. 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.
   3. Collect the cells with 5-10 mL of medium and add to a conical tube.
   4. Count the cells manually with a hemocytometer using standard procedures.
4. 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.
5. Grow the cells in the culture medium under standard conditions.

2. Pulsing cells with IdU

1. 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.
2. After pulsing with IdU for two population doublings, harvest the cells for imaging.
   1. 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

1. 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

1. 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).
2. 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.
3. 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

1. 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.
2. 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.
3. Add 60 µL of IdU antibody dilution on the top of the coverslips. Incubate the coverslips for 1 h at 37 °C.
   1. 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.
4. 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.
5. 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.
6. Aspirate the secondary antibody. Return the coverslips back to the 24-well plate, and wash four times with 0.2% PBST.
7. 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.

Representative Results

Figure 1: IdU labeling schematic. Cells are pulsed with IdU for two population doublings. If any drug treatment is needed, it should be administered after the first population doubling in the presence of IdU.

Figure 2: Comparison of the foci signal obtained after pulsing with IdU, BrdU, or CldU. Representative images of cells pulsed with three different thymidine analogs. The DAPI, GFP (representing Alexa-488-labeled IdU foci), and merged channels are shown. Scale bar: 10 µm.

Figure 3: Increased IdU foci in OVCAR3 cells caused by replication stress. (A) Representative IdU and DAPI-stained nuclei images and (B) quantification of the cells treated with 0.5 mM hydroxyurea for 24 h. **** p < 0.0001 (Mann-Whitney test). Scale bar: 10 µm.

Materials

Name	Company	Catalog Number	Comments
3% Paraformaldehyde (PFA)	Fisher Scientific	NC0179595	10 g sucrose + 100 mL 10X PBS + water to make volume to 925 mL. Add 75 mL 40% Methanol free PFA, mix, and make aliquots of 50 mL before storage Storage: Store in -20 °C
5-iodo-2'-deoxyuridine (IdU)	Sigma Aldrich	I7125-5G	MW = 354.10 g/mol.For 10 mM stock: dissolve 3.541 mg IdU to 1 mL 1 N liquid ammonia
Anti-BrdU antibody	BD Biosciences	347580	Storage: Store in 4 °C
Anti-mouse Alexa Fluor Plus 488 secondary antibody	Thermo Scientific	A32766	Light sensitive - keep in dark
Bovine Serum Albumin (BSA)	Sigma Aldrich	A7906-100G	Made by adding specific mass to volume of PBS Storage: Store in 4 °C
Circular Cover Glass	Electron Microscopy Sciences	72230-01
OVCAR3	ATCC	HTB-161	Growth Media: RPMI supplemented with L-glutamine, 0.01 mg/mL bovine insulin; fetal bovine serum to a final concentration of 20% and 1X Pen Strep Storage: Freezing Media: growth media + 5% DMSO and stored in -80 °C
Poly-L-Lysine solution	Sigma Aldrich	P4832-50ML	Storage: Store in 4 °C
ProLong Diamond Antifade Mountant with DAPI	Thermo Scientific	P36962	Storage: Store in 4 °C
Trypsin-EDTA, 0.25%	Genesee Scientific	25-510	Storage: Store in 4 °C
Water, sterile-filtered	Sigma Aldrich	W3500-6X500ML	Storage: Store in 4 °C