Synergy reader

RNA was isolated from whole tumors harvested at day 21 post-tumor challenge from no therapy and therapy-treated OB-RES and DIO mice (n = 3–8/group). RNA concentration was quantitated using a Take3 micro-volume plate in conjunction with a Synergy reader (BioTek; Winooski, VT, USA). Samples were processed using the nanoString Mouse PanCancer Immune Profiling Kit (nanoString Technologies Inc.; Seattle, WA, USA). Data analyses were performed using nSolver™ Analysis Software (nanoString Technologies, Inc.), by comparing therapy-treated groups to their respective no therapy controls. Due to the exploratory nature of this study, an unadjusted p value less than 0.05 was used to screen for differentially expressed genes. Differentially expressed T cell related genes (listed in Supplemental Table S1) were queried using the Reactome Database [33 (link)].

CXCL1, IL-6 and GM-CSF were measured by ELISA kits purchased from eBioscience. The optical density (OD) was read at 450 nm using a BioTek synergy reader (BioTek).

An amount of 10 µL of sample, at the final concentrations of 10, 25, and 50 µg/mL HHs, was mixed with 140 µL FRAP solution (0.83 mM TPTZ and 1.66 mM FeCl₃ × 6H₂O in 0.25 M acetate buffer, pH 3.6). After 30 min of incubation at 37 °C, the absorbance at 595 nm was measured with the Synergy reader (Biotek). The values were extrapolated by a Trolox (Sigma-Aldrich) standard curve.

Cells were collected in lysis buffer (100 mM Tris pH 7.6, 5 mM EDTA, 1 mM NaN3 and 0.1% peroxide-free Triton-X100). Lysates were complemented with 0.6 U/mL glutathione reductase, 0.2 mM nicotinamide adenine dinucleotide phosphate hydrogen, 3 mM reduced glutathione and 200 μM of the substrate cumene hydroperoxide. NADPH turnover was measured on an BioTek Synergy reader at 340 nm over 10 min at 37° C. Enzymatic activity was calculated after subtracting absorbance decay obtained from buffer without cell lysates by using NADPH extinction and by normalizing to total protein content [40 (link)].

The 504-bp upstream region of nimB was cloned into NheI and SacI sites of pDSW1728 vector to generate PnimB^G::mCherryOpt or PnimB^T::mCherryOpt from R20291 or CD196 respectively. Constructs and the empty vector control were conjugated into various C. difficile strains. Strains bearing the reporter construct and empty vector were grown to OD₆₀₀ nm ~ 0.3, from overnight cultures. Each culture was either treated with DMSO control, metronidazole (2 µg/ml), metronidazole (2 µg/ml) plus hemin (5 µg/ml) or hemin alone (5 µg/ml) and incubated anaerobically for 1 h. Samples were then fixed as described previously^{41 (link)} and fluorescence measured in BioTek Synergy reader at excitation of 554 nm and emission of 610 nm. The fluorescence value for each sample was then normalized based on their culture densities (OD₆₀₀ nm values). Primers and strain constructs used are listed in Supplementary Data 2 and 3.