An Immunofluorescence-Based Method to Quantify Replication Stress in Cancer Cells
Published: April 30, 2024
Abstract
Source: Ramakrishnan, N. et al., Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence. J. Vis. Exp. (2023)
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 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 …
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…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
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Cite This Article
An Immunofluorescence-Based Method to Quantify Replication Stress in Cancer Cells. J. Vis. Exp. (Pending Publication), e22136, doi: (2024).