FxCycle™ Far Red Stain

Protocol tips

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- cells were fixated with either 70% ethanol (Cyclin A, Aurora A and Plk1) or first 1% formaldehyde (1 h on ice) followed by 70% ethanol

Upstream tips
- cells were fixated with either 70% ethanol (Cyclin A, Aurora A and Plk1) or first 1% formaldehyde (1 h on ice) followed by 70% ethanol

Publication protocol

For analysis of protein expression in different cell cycle phases, cells were fixated with either 70% ethanol (Cyclin A, Aurora A and Plk1) or first 1% formaldehyde (1 h on ice) followed by 70% ethanol (Cyclin B1, p21 and Chk2) and stained with rabbit anti‐phospho‐Histone H3(Ser10) (06‐570, Millipore) and mouse anti‐Cyclin B1 (sc‐245, Santa Cruz), anti‐Chk2 (DCS 270.1 (Sørensen et al., 2003)) or anti‐Plk1 (37‐7000, Invitrogen/Life Technologies), alternatively mouse anti‐phospho‐Histone H3(Ser10) (05‐806, Millipore) and rabbit anti‐Cyclin A (sc‐751, Santa Cruz), anti‐Aurora A (ab1287, Abcam) or anti‐p21 (sc‐756, Santa Cruz). Mouse anti‐phospho‐Histone H2AX(Ser139) (05‐636, Millipore) and rabbit anti‐phospho‐BRCA2(Ser3291) (AB9986, Millipore) were used for analysis of DNA damage and CDK activity, respectively. Antibody staining was performed as described previously (Tkacz‐Stachowska et al., 2011). Barcoding (Krutzik and Nolan, 2006; Tkacz‐Stachowska et al., 2011) was used to eliminate variation in antibody staining between individual samples. For barcoding, four individually fixed samples were stained with different concentrations (0.125, 0.031, 0.0062, and 0.00078 ng/μl) of Pacific Blue succinimidyl ester (Invitrogen) for 30 min in the dark at room temperature, and subsequently mixed into one tube. The mixed cells were then stained with antibodies and the DNA‐stain FxCycle Far Red (Invitrogen). For each barcoding experiment an aliquot of the mixed cells was analyzed without any antibodies to check the need for compensation of the Pacific Blue signal, and an aliquot was stained with secondary antibodies only (no primary antibodies) to correct for background staining (see Figure S1). When repeating individual experiments, the concentrations of Pacific Blue were alternated between the treatment conditions to avoid any potential influence on the results by the Pacific Blue staining. For analysis of G2 checkpoint activation, cells were stained with rabbit anti‐phospho‐Histone H3(Ser10) (06‐570, Millipore) and the DNA stain Hoechst 33258 (1.5 μg/ml) (Sigma–Aldrich). Flow cytometry analysis was performed on a LSRII flow cytometer (BD Biosciences) using Diva software. Error bars represent standard errors of mean from at least 3 independent experiments.

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