Sybr green dna stain

To analyze DSB, cells were plated at 0.2 x 10⁶ cells per well in 6‐well plates the day before treatment. PIT was administered at the indicated doses 1 h prior to 10 Gy irradiation. After 20 h, cells were mixed with Comet LM agarose (Bio‐Rad) and single‐cell electrophoresis was performed on CometSlides (Trevigen). Slides were fixed, dried, stained with SYBR Green DNA stain (1/10 000×; Life Technologies), and imaged on an Axiovert 40 with a 20X Plan‐NeoFluar objective and an AxioCam monochromatic fluorescence camera. Images were analyzed using a Comet assay macro for ImageJ software (http://www.med.unc.edu/microscopy/resources/imagejplugins-and-macros/comet-assay).

IEM were labelled with SYBR Green DNA stain [Life Technologies, 1/100 000], anti-IgA-APC/anti-IgG-APC/Cy7, and analysed by flow cytometry to determine proportion [%] of bacteria coated with antibodies in the gut. Circulating antibodies to commensal species were determined by incubation of plasma [1/10 in 0.1% BSA, 0.5 ml] with 1 × 10⁵ bacteria [30 min], followed by centrifugation [12 000 g, 10 min] and staining as for IEM [or isotype controls for each sample]. Intact microbes were gated according to SYBR Green, and ratio of geometric mean fluorescence intensity of staining for test sample vs isotype control was used as measure of antibody titre. Plasma IgG antibodies to viruses were measured using ELISA kits from Abcam according to instructions.

Reference chemicals benzo[a]pyrene (BaP, B1760), ethyl methanesulfonate (EMS, M0880), 1,2-dimethylhydrazine hydrochloride (DMH, D161802), etoposide (Etop, E1383), phenformin hydrochloride (Phen HCl, PHR1573), potassium bromate (KBr, 309087), glycidamide (GA, 04704), and ethyl nitrosourea (ENU, N3385) were purchased from Sigma Aldrich (St. Louis, MO). Test chemical stock solutions were prepared in anhydrous DMSO (Sigma Aldrich, St. Louis, MO) or in water.
Cell recovery solution (Corning, Tewksbury, MA) and human tumour dissociation kit (130-095-929, Miltenyi Biotec, Bergisch Gladbach, Germany) were used for the purpose of 3D tissue dissociation as previously described [31 (link)].
Comet assay kit consisting of low-melt agarose (4250-050-02, 1% low melting agarose in PBS), cell lysis solution (4250-050-01), pre-coated 2-gel agarose comet slides (CometSlide™ 4250-200-03), and electrophoresis apparatus (4250-050-ES) were sourced from RnD systems (Minneapolis, MN). Low-melt agarose was reused up to 10 times after melting. TE buffer was obtained from Promega (Madison, WI). Alkali DNA unwinding (300 mM NaOH, 1 mM EDTA) and electrophoresis solutions (200 mM NaOH, 1 mM EDTA) were prepared fresh for use on the day of experiment. SYBR Green DNA stain was procured from Thermo Fisher Scientific (Waltham, MA).