Alexa 647 azide

For cell cycle analysis, cells were trypsinized, fixed with ice-cold 70% ethanol overnight at 4 °C, treated with propidium iodide (PI) solution (50 µg/ml PI, 50 µg/ml RNaseA, and 0.1% Triton X-100, 1% FBS in PBS) for 30 min and acquired using BD FACSCanto. Results were analysed using FlowJo software. For EdU detection, Alexa 647 was covalently linked to EdU by click chemistry reaction for 45 min in the dark (Alexa 647-azide (Thermo Fisher, A10277), 10 mM Sodium ascorbate, 2 mM CuSO₄ in 1× PBS).

Spheroids were fixed in 4% paraformaldehyde for 1–2 h, preserved overnight in a 30% sucrose solution, embedded in OCT (Sakura), and sectioned into 10 µm sections using a cryostat NX70 (Thermo Fisher). For immunofluorescence, sections were blocked and permeabilized using a 5% bovine serum albumin (Sigma Aldrich) in 0.25% Triton solution for 2 h and incubated with primary antibody overnight at 4°C (Supplemental Table 4, http://links.lww.com/HEP/I55). For labeling S-phase, 10 µM EdU (Sigma Aldrich/Merck) was added into the culture medium and revealed by incubation for 30 minutes in staining solution (0.1 M Tris pH 7.5, 2 mM CuSO₄, 5 µM Alexa 647-azide (Thermo Fisher), 100 mM ascorbic acid). Nuclei were counterstained using DAPI (Thermo Fisher). Nuclear p21 intensity quantifications were conducted using FIJI software by measuring EdU and p21 channel intensities using a binarized mask based on the DAPI channel.

Antibodies were purchased from the following sources: Cdt1 (Cat# 8064, at least two different lots were used in this study), Chk1 (Cat# 2345), phospho-Chk1 S345 (Cat# 2341), Cyclin E1 (Cat#4129), MAPKAPK-2 (Cat#), Phospho-MAPKAPK-2 T334 (Cat#3007), phospho-Histone H2A.X Ser139 (Cat#9718) from Cell Signaling Technologies; hemagglutinin (HA) (Cat#11867423001) from Roche; Geminin (Cat#sc-13015), Cdc6 (Cat#sc-9964), MCM6 (Cat#sc-9843), Cyclin A (Cat#sc-596), Cyclin B1 (Cat#sc-245) and CDK2 (Cat#sc-163) from Santa Cruz Biotechnology; MCM4 (Cat#3728) from Abcam. MCM2 antibody (Cat#A300-191A) used for co-immunoprecipitation experiment was purchased from Bethyl Laboratories. MCM2 antibody (BD Biosciences, Cat#610700) was used for analytical flow cytometry. Serum to detect CDK1 was a gift from Y. Xiong (University of North Carolina), and MPM2 antibody was a gift from R. Duronio [82 (link)] (University of North Carolina). The phosphospecific Cdt1 antibody was described in Chandrasekaran et al [17 (link)].; the third and fourth test bleeds are active for Cdt1 immunoprecipitation. Alexa 647-azide and Alexa-488-azide used in flow cytometry analyses was purchased from Life Technologies, and secondary antibodies for immunoblotting and immunofluorescence were purchased from Jackson ImmunoResearch.

For analytical flow cytometry, EdU (Santa Cruz Biotechnology) was added to 10 μM 30 min before collection with trypsin. Cells were permeabilized with 500 μl cytoskeletal (CSK) buffer supplemented with 0.5% Triton X-100 and protease and phosphatase inhibitors as used above for immunoblotting, then fixed with PFA and subjected to antibody staining and EdU detection as described here dx.doi.org/10.17504/protocols.io.bba8iihw. Samples were analyzed on an Attune NxT flow cytometer (Life Technologies). Flow cytometry data were evaluated using FCS Express 7.0 software (De Novo). Cells were gated on FS(A) x SS(A) plots, and singlets were gated using DAPI(A) x DAPI(H) plots. Control samples were prepared as described previously (Matson et al, 2017 (link)). Control samples were treated the same as experimental samples by incubating with azide (either 647 or 488) and the appropriate secondary antibody. The following antibody/fluorophore combinations were used: (1) MCM (measuring origin licensing): Alexa 647-azide (Life Technologies), primary: Mcm2 (Cat. no. 610700; BD Biosciences), secondary: donkey anti-mouse-488 (Jackson ImmunoResearch), DAPI. (2) γ-H2AX (measuring DNA damage): Alexa 488-azide (Life Technologies), primary: γ-H2AX (Cat. no. 9718; Cell Signaling Technologies), secondary: donkey anti-rabbit 647 (Jackson ImmunoResearch), DAPI (Cat. no. D1306; Life Technologies).