HT immunofluorescence for γH2AX DNA damage

High-throughput immunofluorescence for γH2AX was performed in a 384 well plate and images were captured with an Opera High Content Screening System as described in the HT microscopy section. First, frozen erythrocyte lysis buffer processed blood samples were thawed quickly and resuspended in complete RPMI media. Cells were attached to the wells (30,000–150,00 cells/well of clear-bottom black-walled 384-well plates (CellCarrier-384 Black Optically Clear Bottom plates Cat No 6007550) which had been precoated with 0.001% weight/volume (poly)L-lysine solution (Sigma P8920-100 mL) for 30 min at 37 °C. The wells on the outer edge of the plate were not used. The cells were incubated for 37 °C for no more than 4 hours before fixation. The cells were then fixed for 10 min by adding formaldehyde to the media so that the final concentration of formaldehyde was 4%. The cells were washed 3X with TBS (Tris-Buffered Saline) 5 min, permeabilized with 0.5% Triton X-100 in TBS 10 min, washed 3X with TBS 5 min, and then blocked by adding FBS. The plate was incubated for 2 hr at room temperature. After the blocking, the primary antibody (1:1000 Cell Signaling Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb Cat No 97185) in 5% BSA in PBS was added, and the plate was incubated overnight at 4 °C. The next day, the primary antibody solution was removed, the wells were washed 3X with TBS 5 min, the secondary antibody (Invitrogen Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Cat No A11008) diluted in 5% BSA in PBS was added, the plate was incubated 1 hr at room temperature, the wells were washed 3X with TBS 5 min, the wells were washed 1X with TBS containing 1 μg/mL DAPI (4′,6-diamidino-2-phenylindole) to stain the nuclei, the plate was washed 1X with TBS 5 min, the TBS was removed and 50 μL of Mowiol solution (10 g mowiol (polyvinyl alcohol; Calbiochem Cat no 475904), 25 mL glycerol, 25 mL H₂0, 12 mL 0.2 M Tris HCl pH 8.5, and 2.5% w/v DABCO (1,4-Diaza [2.2.2] bicyclooctane; Sigma-Aldrich Cat no D27802-25G)) was added. The plates were then covered with aluminum foil lids to block light and stored at 4 °C in the dark until imaging as described in the HT microscopy section.