Cells were treated with AZD1775 and/or olaparib for 48 hours in poly-D-lysine coated chamber slides (Sigma-Aldrich, St Louis, MO). Cells were then fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.2% Triton X in PBS for 10 minutes. Cells were incubated in blocking solution (5% milk in 0.05% Triton X-PBS) for 30 minutes followed by an overnight incubation in anti-Rad51antibody (Cell Signaling Technology) at a dilution of 1:500. After several washes, Alexa Fluor 488 conjugated anti-rabbit antibody (1:500) was applied for 1 hour. ProLong Gold Antifade Mountant with DAPI (Life Technologies, Waltham, MA) was used for mounting. Images were acquired using an inverted epifluorescence microscope at 100X magnification.
Poly d lysine coated chamber slides
Manufactured by Merck Group
Poly-D-lysine coated chamber slides are a type of laboratory equipment used for cell culture. They provide a positively charged surface that promotes the attachment and growth of adherent cells.
Automatically generated - may contain errors
3 protocols using poly d lysine coated chamber slides
1
Quantifying DNA Damage Response to AZD1775 and Olaparib
2
Spinal Cord Injury Neurosphere Culture
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Animals were sacrificed for control culture or one week after SCI. Spinal cord cells were dissociated and neurosphere cultures were established as described (Meletis et al., 2008 (link)). All cells isolated from one spinal cord were plated in 10 cm culture dishes. First, neurospheres were harvested after 2 weeks in culture and then were dissociated into single cells for passage or differentiation. Approximately 100,000 cells per animal were plated in a 10 cm culture dish for the next generation of neurospheres, and all the new neurospheres (second, third and fourth generations) were harvested after one week in culture. Dissociated primary neurospheres, approximately 50,000 cells/well, were plated in poly-d -lysine-coated chamber slides (Sigma) for differentiation with growth factors-free medium supplemented with 1% fetal bovine serum. Two to four independent experiments per group were performed.
3
Senescence Induction in Cells
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