RPP631 cells were incubated in the presence of 100nM YKL-5–124 for 48–72 hr. EdU was pulsed during the last 30 minutes of YKL-5–124 treatment. Treated cells were pre-extracted using 0.5% Triton in CSK buffer (10 mM Hepes, 300 mM Sucrose, 100 mM NaCl, and 3 mM MgCl2, pH = 7.6) for 10 minutes, and fixed with paraformaldehyde (4%) for 30 minutes. Cells were then rinsed 3 times with PBS and blocked (2% glycine, 2% BSA, 0.2% gelatin, and 50mM NH4Cl in PBS) overnight at 4 °C for further staining. EdU was tagged with Alexa Fluor 647 picolyl azide through Click reaction (Thermo Fisher, C10640) before immunofluorescent labeling of target proteins, for which the antibodies are mouse anti-PCNA (Santa Cruz Biotech., sc56, 1:1000), goat anti-mouse (Alexa Fluor 750 conjugated, Thermo Fisher, A-21039, 1:5000), and rabbit anti-MCM2 (Alexa Fluor 647 conjugated, Abcam ab223403, 1:500). Fixed cells were mounted onto microscope glass for STORM imaging in freshly mixed imaging buffer (1 mg/mL glucose oxidase (Sigma-Aldrich, G2133), 0.02 mg/mL catalase (Sigma-Aldrich, C3155), 10% glucose (Sigma-Aldrich, G8270), and 100 mM cysteamine (MEA, Fisher Scientific, BP2664100).
C10640
Manufactured by Thermo Fisher Scientific
The C10640 is a high-performance digital CCD camera designed for scientific and industrial imaging applications. It features a large 6.45 mm x 4.84 mm sensor with 1024 x 768 active pixels and a high quantum efficiency. The camera is capable of producing high-quality images with low noise and high dynamic range.
Automatically generated - may contain errors
Lab products found in correlation
G2133, by Merck Group (1 mentions)
C3155, by Merck Group (1 mentions)
Bp2664100, by Thermo Fisher Scientific (1 mentions)
Ab6154, by Abcam (1 mentions)
Ab79398, by Abcam (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
A11036, by Thermo Fisher Scientific (1 mentions)
Dead cell apoptosis kit with annexin 5 fitc, by Thermo Fisher Scientific (1 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using c10640
1
STORM Imaging of Cell Cycle Proteins
2
Chromatin-bound Replisome Visualization
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Cells were immediately permeablized and fixed after EdU labeling. In brief, cells were extracted with CSK buffer (10 mM Hepes, 300 mM Sucrose, 100 mM NaCl, 2 mM MgCl2, and 0.5% Triton X-100, pH = 7.4) for 10 minutes to remove replisome proteins that are unbound to chromatin, followed by fixation with paraformaldehyde (PFA, 4% in PBS) for 30 minutes24 (link)25 (link). Cells were then washed twice with blocking buffer (2% glycine, 2% BSA, 0.2% geltin, and 50 mM NH4Cl in PBS) and blocked for 1 hour at room temperature or overnight at 4 °C for further immunofluorescence staining.
Nascent DNA staining was performed by tagging Alexa Fluor 647 picolyl azide onto EdU via the ‘click’ reaction26 (link) (Click-iT chemistry, thermoFisher C10640). RPA, PCNA, and MCM were stained with validated monoclonal antibodies27 (link)28 (link)29 (link). In detail, RPA was immunostained with a rabbit monoclonal against RPA70 (Abcam, ab79398) followed by secondary immunostaining with goat-anti-rabbit conjugated with Alexa Fluor 568 (ThermoFisher, A11036); PCNA and MCM were immunostained with Mouse monoclonal against PCNA (Abcam, Ab29) and MCM5 (Abcam, ab6154), respectively, followed by secondary immunostaining with goat-anti-mouse conjugated with alexa Fluor 488 (ThermoFisher, A11029).
Coverslips were mounted onto a microscope microfluidics chamber for SR imaging.
Nascent DNA staining was performed by tagging Alexa Fluor 647 picolyl azide onto EdU via the ‘click’ reaction26 (link) (Click-iT chemistry, thermoFisher C10640). RPA, PCNA, and MCM were stained with validated monoclonal antibodies27 (link)28 (link)29 (link). In detail, RPA was immunostained with a rabbit monoclonal against RPA70 (Abcam, ab79398) followed by secondary immunostaining with goat-anti-rabbit conjugated with Alexa Fluor 568 (ThermoFisher, A11036); PCNA and MCM were immunostained with Mouse monoclonal against PCNA (Abcam, Ab29) and MCM5 (Abcam, ab6154), respectively, followed by secondary immunostaining with goat-anti-mouse conjugated with alexa Fluor 488 (ThermoFisher, A11029).
Coverslips were mounted onto a microscope microfluidics chamber for SR imaging.
3
Evaluating Cell Proliferation and Survival
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Cells were seeded in collagen gels and allowed to grow for 10 days with or without DDR1i (2 µM). For EdU proliferation assay, EdU was added on day 10 and gels were allowed to grow for two more days and fixed for analysis on day 12. EdU fluorescence detection was performed according to the manufacturer’s protocol (C10640, Thermo Fisher). The percentage of EdU+ cells per organoid was analyzed in 10 organoids of each group. For cell cycle analysis, cells were harvested on day 10 and labeled with DRAQ5 membrane permeable dye (65-0880-96, Invitrogen) at a concentration of 5 µM and analyzed using a flow cytometer (BD LSRII at the Tufts Flow Cytometry Core). Analysis of cell cycle phases was performed on FlowJo software v10.7 using the Dean-Jett-Fox model. For dye dilution analysis, CellTrace CFSE cell proliferation kit for flow cytometry was used (C34570, Thermo Fisher) according to manufacturer protocol. Cells were labeled with 5 µM CFSE immediately before embedding in collagen gels, and isolated and analyzed by flow-cytometry (as above) after 3 days in culture. For Annexin V assay, Dead Cell Apoptosis Kit with Annexin V FITC was used (V13242, Thermo Fisher) according to manufacturer protocol. For cell cycle, dye dilution and annexin V analyses, 3 samples were individually cultured, treated, isolated and analyzed in each treatment group.
4
Ovarian Cell Proliferation Assay
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Adult female ovaries were dissected in 1× PBS, transferred to a microfuge tube containing 10 mM EdU in 1× PBS, and kept at room temperature on a shaker for 1 h. Ovarioles were then dissociated, fixed and stained with primary and secondary antibodies as described above. The EdU detection reaction was performed according to the manufacturer's manual (Thermo Fisher Scientific, C10640).
5
Quantifying Cell Proliferation with EdU Assay
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Proliferation was quantified using Click‐iT™ Plus EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 647 dye (Thermofisher, C10640). Cells were transfected with siRNAs in eight‐well chamber slides at a density of 1 × 104 cells per well (48 h) or 3.75 × 103 cells per well (7 days). After 48 h or 7 days of incubation, cells were exposed to 10 μM of 5‐ethynyl‐2′‐deoxyuridine (EdU) for 4 h at 37°C and then fixed with PFA 4%. EdU labelling was done according to manufacturer's instructions (Life Technologies, C10640) and nuclei were stained with Hoechst 33342. Images were acquired using a confocal microscope and quantification was done using the Cell Counter Plugin in ImageJ to manually tag and count stained cells for each color channel and apoptotic bodies.
6
Neurogenesis Quantification in Drosophila
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Third-instar larval brains were dissected out in Shields and Sang M3 insect medium and incubated in fresh M3 medium containing 10% FBS and 40 µM EdU at RT for 1 h. The medium containing EdU was removed, and the brains were rinsed thrice with PBS. The brains were fixed with 4% PFA in in PBT (0.1% Triton X-100) at RT for 20 min and blocked with 0.1% BSA in PBT (0.3% Triton X-100). The Click-iT reaction was performed according to the manufacturer’s instructions (Life Technology, C10640), and the slides were then immunostained for E-cadherin to label neuroepithelial cells and cyclin-E and mounted in Vectashield mounting medium.
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