*Appendix E.1. Single Antibody (AB) Staining*

Single antibody staining uses one primary antibody and one secondary antibody to stain the cells. The cells are grown on square coverslips in p35 culture dishes. Paraformaldehyde (PFA, from Electron Microscopy Sciences, PA, USA), Triton X-100 (from American Bioanalytical, MA, USA), and bovine serum albumin (BSA, from Gold Biotechnology, MO, USA) are previously prepared with desired concentrations in 1X PBS.

Day 1:


$$\mathcal{V}\_{\text{AB}}(\mu\text{L}) = \frac{\mathcal{V}\_{\text{total}}}{\text{dilution factor}} = \frac{75\mu\text{L} \times \mathcal{N}\_{\text{sample}} + \text{30}\mu\text{L}}{250} \tag{A1}$$

$$V\_{BSA} = V\_{total} - V\_{AB} \tag{A2}$$

where VAB is the volume of antibody from the stock, Vtotal is the volume of the final solution, VBSA is the volume of the 3% BSA as a solvent. The volume of the final solution applied to each coverslip for antibody staining is 75 µL. There is an extra volume of 30 µL in case of bubble formation inside the solution during pipetting and mixing.


Day 2:


#### *Appendix E.2. Double Antibody Staining*

Double staining (DS) uses two primary antibodies and two secondary antibodies with different fluorescence emission ranges to stain two different parts of a cell. The procedures of double antibody staining are the same as the single antibody staining except following modifications of the step (8) and the step (17) to be (26) and (27), respectively:

(26) Prepare AB solution with two primary ABs. For example, anti-phospho-histone H2AX and cleaved caspase-3 antibody need to be diluted in 3% BSA with a ratio of 1:250 and 1:400, respectively. The amount of antibody can be calculated as follows:

$$V\_{AB1}(\mu L) = \frac{V\_{\text{total}}}{\text{dilution factor 1}} = \frac{75\mu L \times N\_{\text{sample}} + 30\mu L}{250} \tag{A3}$$

$$V\_{\text{AB2}}(\mu\text{L}) = \frac{V\_{\text{total}}}{\text{dilution factor 2}} = \frac{75\mu\text{L} \times \text{N}\_{\text{sample}} + 30\mu\text{L}}{400} \tag{A4}$$

$$V\_{\rm BSA} = V\_{\rm total} - V\_{\rm AB1} - V\_{\rm AB2} \tag{A5}$$

where VAB1 and VAB2 are the volumes of the two antibody solutions needed.

(27) Prepare AB solution with two secondary ABs. For example, both Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit need to be diluted in 3% BSA with the ratio of 1:400. The amount of each antibody can be calculated in the same method as Equations (4) and (5) with a dilution factor of 400.

#### *Appendix E.3. S Phase Cell Staining with Click-iT EdU Alexa Fluor 647 Imaging Kit and Single AB*

Cells are grown on square coverslips in p35 culture dishes. PFA, Triton X-100, and BSA are previously prepared with desired concentrations in 1X PBS. Different stock solutions are previously prepared according to the manufacture protocol [48].


#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
