E. All screens were carried out at passage 105. Cells were tested for mycoplasma by PCR prior to seeding and infection. U2OS cells had been seeded using a MicroFill (Biotek) in 384-well black, clear bottom Bifemelane Epigenetics plates (Greiner) at a density of 300 (shRNA) cells/well in 50 L of media, and permitted to attach overnight at 37 inside a five CO2 atmosphere. For shRNA screens, the media was exchanged the following day to finish media with eight g/mL polybrene using a JANUS workstation (PerkinElmer). Virus infection was carried out on an EP3 workstation (PerkinElmer) with 1.5 L of hightiter retrovirus. All plates had two wells F16 manufacturer infected with 1.five L of manage virus with shRNA directed against H2AX. Plates have been centrifuged inside a swinging-bucket rotor at 2250 rpm for 30 minutes following infection and returned to the incubator overnight. The plates have been then chosen with 2.five g/mL puromycin for 48 hours, and permitted to proliferate in total media for an additional 48 hr, with media exchanges carried out around the JANUS or RapidPlate (Qiagen) liquid handling workstations. Eight wells in each plate had been not chosen with puromycin. For small molecule testing, cells were plated at 500 cells/well in 384-well plates. The dayAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptNature. Author manuscript; offered in PMC 2013 December 13.Floyd et al.Pageafter plating, small molecules at distinct concentrations in 100 nL DMSO had been pin transferred to cells using a CyBio robot, and cells had been propagated for 16 hr. For each small molecule and shRNA screens, 4 plates were made in replicate for the timepoints outlined beneath. Four wells were left untreated in every plate, and received 25 mM caffeine in total media 1 hr before irradiation. All plates have been treated with 10 Gy of 667 keV Xrays from a 137Cs source inside a Gammacell irradiator (Atomic Power of Canada, Ltd). A 0 hr control plate was not irradiated. The plates have been returned towards the incubator and fixed with four.4 w/v paraformaldehyde in phosphate-buffered saline (PBS) at 1, 6, and 24 hr postirradiation. Plates had been stored in PBS at four before staining. Fixed plates were washed 3 times with PBS and blocked with 24 L of GSDB (0.15 goat serum, 8.33 goat serum, 120 mM sodium phosphate, 225 mM NaCl) for 30 minutes. The 0, 1, and 6 hr plates have been incubated with 1:300 dilutions in GSDB of primary mouse monoclonal anti-H2AX (Ser 139), and rabbit polyclonal anti-pHH3 antibody. For the 24 hr plates, we substituted 1:300 rabbit polyclonal anti-cleaved Caspase 3 for the pHH3 antibody. All plates had been incubated overnight at four , washed, and stained using a secondary antibody mix containing 10 g/mL Hoescht 33342, 1:300 goat anti-mouse polyclonal-Alexa Fluor 488, and goat anti-rabbit polyclonal-Alexa Fluor 555 in GSDB. After a second overnight incubation at 4 , the plates have been washed 3 times in PBS and stored in 50 L/well 50 M Trilox (Sigma) in PBS at four . Imaging and image evaluation Plates were allowed to equilibrate to space temperature for 30 min and imaged on a Cellomics ArrayScan VTI automated microscope with a 20x objective. The acquisition parameters were exactly the same for each and every shRNA or chemical library. Six fields per well were imaged, with 3 channels/field (DAPI, fluorescein and rhodamine) for any total of 18 acquired pictures per effectively. Photos had been segmented and analyzed with CellProfiler cell image evaluation software program (Carpenter et al., Genome Biology 2006, 7, R100). The imaging pipeline made use of to segment the images is av.
