Facscelesta cell analyzer

To assess IgM deposition, 1 × 10⁷R. parkeri or R. australis bacilli were fixed in 4% PFA solution. After washing with PBS-glycine, bacteria were incubated with 30% of NHS, IgM-dplHS, or PBS for 1 hr at RT or overnight at 4°C. To assess both human IgM and C1q deposition on Rickettsia bacilli, all samples were treated with rabbit anti-human C1q (Thermofisher), Alexa Fluor-546-conjugated donkey anti-rabbit-IgG (Invitrogen) and AlexaFluor-488-conjugated goat IgG anti-human IgM (Invitrogen). Mouse IgM was measured as above but with C57BL6/J serum and AlexaFluor-488-conjugated anti-mouse IgM (clone AF6–78, Invitrogen). Fluorescence was assessed on a FACSCelesta Cell Analyzer (BD Biosciences) using fluorescein isothiocyanate (FITC) and phycoerythrin (PE) parameters and performed as previously described (Chan et al., 2011 (link); Riley et al., 2014 (link)). All samples were analyzed with FloJo software (Tree Star).

U2OS cells stably transfected with DR-GFP plasmid was a kind gift from Dr. Guang Peng at MD Anderson Cancer Center and used for homologous recombination (HR) assay, as described previously [22 (link), 23 (link)]. Briefly, these cells were transfected with the endonuclease pCBASceI plasmid (2 μg) obtained from Addgene (#26477) using Lipofectamine 3000 (10 μl) for 24 hours in a 6-cm dish. Cells were then seeded into 6-well plates and incubated for another 24 hours, when cells were exposed to the drugs for 24 hours and GFP positive cells were determined by FACS (FACSCelesta™ Cell Analyzer, BD Bioscience, San Jose, CA). Transient transfection was used to similarly assay HR in A2780 cells, as previously reported [24 (link)]. Briefly, cells in 6-well plates were co-transfected with plasmids from Takara Bio (0.25 μg mCherry-C1, #632524) and Addgene (1 μg pDR-GFP, #26475; 1.5 μg pCBASceI) using Lipofectamine 3000 (3.6ul). The next day, cells were incubated with platinum compounds and then subjected to FACS analysis 24 hr later. GFP-positive cells were normalized to mCherry-positive cells for transfection efficiency In either tumor model, downregulation of HR inhibits production of GFP and, thereby, correlates with DNA repair.