DDR markers (Stefano Ferrari's protocol)

All centrifugations: 5’ @450g, 4°C. One can use 1.5 ml or FACS tubes for the staining – for staining or pre-extracted cells, I always use FACS tubes because it is easier to decant the supernatant after spinning.

Protocol

1. EdU only: Add EdU to cells of interest to final concentration of 10µM (10mM stock)
2. Incubate 30’@37°C
3. Harvest cells by trypsinisation
4. Wash cells with 1x PBS (Eppendorf centrifuge 5810R: 450xg)
5. RPA only: pre-extract by incubating the cells in 100 µl 0.2% Triton X-100/PBS for 10’ on ice
6. RPA only: Add PBS to 1 ml and spin down
7. Incubate cells in 300 µl 4% FA/PBS for 15’ at RT
8. Add 1% BSA/PBS to 1 ml and spin down
9. Wash with 1 ml 1% BSA/PBS
10. Decant supernatant, resuspend cells in 1 ml 1% BSA/PBS, store @4°C – PROCESS SAMPLES THE SAME DAY OR THE DAY AFTER
11. Split cells into sample tubes, in case you do multiple staining on the same sample. Spin down
12. Resuspend cells in 100µl saponin buffer containing 1° antibodies:
    Mouse α-γH2AX 1:2000
    Rabbit α-γH2AX 1:500
    Mouse α-MPM2 1:1000
    Mouse α-RPA 1:50
    Rabbit α-pH3 1:133
    Using FACS tubes; decant the supernatant and a little bit of buffer remains in the tube. In this case, I add 50µl of buffer containing 2x antibody.
13. 2hrs @RT
14. Add saponin buffer to 0.5 ml and spin down
15. Wash with 0.5 ml saponin buffer
16. Resuspend cells in 50 µl saponin buffer containing appropriate 2° antibodies:
    Alexa 647 1:50 (FACS channel APC) choose colors according to your FACS!!!
    Alexa 750 1:50 (FACS channel APC-Cy7)
    Alexa 488 1:50 (FACS channel FITC)
    Using FACS tubes, I decant the supernatant and a little bit of buffer remains in the tube. In this case, I add 25µl of buffer containing 2x antibody.
17. 30’ @RT in the dark
18. Add saponin buffer to 0.5 ml and spin down
19. Wash with 0.5 ml saponin buffer
    
    
20. EdU only: Resuspend EdU-labelled cells in ClickIT reaction mix (100 µl/sample):
    87.5 µl 1X ClickIT Reaction Buffer or PBS
    2 µl CuSO4
    0.5 µl A488 azide (FACS channel FITC)
    10 µl Buffer Additive (dilute 10x solution in H20, then take 10 µl)
    100 µl final volume
    
    
21. EdU only: 30’ @RT in the dark
22. EdU only: Add 1% BSA/PBS to 1 ml and spin down
23. EdU only: Wash with 1 ml 1% BSA/PBS
24. Resuspend cells in 0.5 ml 1% BSA/PBS containing 0.1mg/ml RNase and 1µg/ml DAPI (FACS channel Violet 1)
25. 15-30’ @RT in the dark… and measure!

Recipes and antibodies

Saponin buffer
10% saponin : BSA/PBS/azide (1:20)

10% saponin Dissolve 10g saponin (Calbiochem, Cat# 558255) in 100ml PBS, pH7.4 Incubate at 37°C until saponin is completely dissolved, sterile filter Store at 4°C

BSA/PBS/azide
Dissolve 2.5g BSA in 500ml PBS and add 0.5ml 1M NaN3, sterile filter Store at 4°C

Antibodies
Mouse α-γH2AX – Millipore # 05-636
Rabbit α-pH3 – Millipore # 06-570
Rabbit α-γH2AX – Cell Signaling #9718
Mouse α-MPM-2 – Millipore # 05-368
Mouse α-RPA – Calbiochem (RPA 34-20) NA19L
ClickIT Kit – Invitrogen EdU Alexa Fluor 488 Flow Cytometry Assay Kit Cat# C10425

Western
pKAP1 S824 – Bethyl labs, Cat A300-767A, total KAP1 – Bethyl labs, Cat A300-274A
pCHK1 S345 – Cell Signaling, rabbit mAb, #2348, total CHK1 – Santa Cruz, mouse, sc-8408

IF
Rad51 – Santa Cruz, (H92) rabbit, sc-8349
53BP1 – Abcam, rabbit, ab21083