Dihydrorhodamine 123

MDMs were detached by incubation with TrypLE™ Express solution (Life Technologies) at 37°C for 10 min. For the analysis of surface markers, monocytes and MDMs were incubated for 20 min with saturating concentrations of monoclonal antibodies against HLA-DR (BioLegend; clone L243), CD86 (BioLegend; clone IT2.2), CD206 (BioLegend; clone 15-2), and CD163 (BioLegend; clone GHI/61). Cells were also stained with dihydrorhodamine 123 and 4-amino-5-methylamino-2',7′-difluorofluorescein diacetate, all from Invitrogen.
For experiments with neutrophils, cells were stained with BD Horizon^TM Fixable Viability Stain 450 (BD Biosciences), followed by the surface monoclonal mouse antihuman-conjugated antibodies: anti-CD15-PE (clone H198) and anti-CD11b-FITC (ICRF44) from BioLegend. Intracellular staining was performed after fixation and permeabilization with Fix/Perm kit (eBiosciences), with the antibodies anti-IL-1β-FITC (clone JK1B-1), anti-IL-6-APC (MQ2-13A5), anti-TNFα-APC (Mab11) from BioLegend, and anti-IL-10-FITC (BT-10) from eBioscience.

ROS production was analysed using a modification of our previously described method [8 (link)]. ELISA plates were pre-coated (2h, 37°C) with/without IgG or F(ab´)₂ at 10 μg/ml. Following washing and blocking of plates with 1%MP/PBS (1h, 37°C), wells were incubated (1h, 37°C) with 100μl patient or control serum (diluted 1 in 50 in media) prior to washing.
Granulocytes (1x10⁶/ml) were incubated (15min/37°C) with ROS indicator Dihydrorhodamine 123 (Invitrogen, 5uM) then 200ul added per well of an ELISA plate either uncoated or coated as described above. Following incubation (1h/37°C), the MFI of green fluorescent rhodamine 123 was determined by flow cytometry.

Reactive oxygen species were analyzed using fluorescent probe dihydrorhodamine 123 (Invitrogen; Thermo Fisher Scientific, IL, United States) applied to frozen heart sections. The sections were mounted and observed with fluorescent microscopy (i50 Eclipse, Nikon, Hamburg, Germany) at a final magnification of 400×. Twenty random fields from a total of five non-consecutive sections per animal were analyzed, and the fluorescence staining was calculated using an image analyzer (Image Pro Premier 9.1, Media Cybernetics, Rockville, MD, United States) and expressed as arbitrary units (AU). Two blinded investigators performed the analysis, and their evaluations were assumed correct if the values were not significantly different. If there was disagreement concerning the interpretation, the case was reconsidered in order to reach a unanimous agreement.

NeuPs or neutrophils were either sorted from the BM or cultured in vitro in the presence of G-CSF for 24 hours at 37 °C. Cells were stained with 5 μM dihydrorhodamine 123 (Invitrogen). N-acetylcysteine (NAC) was treated while staining with DHR123 to show the negative staining. ROS production was stimulated by treating cells for 30 minutes with E. coli (DH5α, multiplicity of infection (MOI) = 10) alone for the sorted cells or with Low-Tox-M Rabbit Complement (Cedarlane) and E. coli (DH5α, MOI = 10) for the cultured cells. Dead cells were excluded with DAPI and stained cells were analyzed on an LSRFortessa (BD Biosciences); data were analyzed using FlowJo software (Tree Star).

Lymphoma cells were incubated at a cell density of 0.5 × 10⁶/mL in complete media containing DMSO or metformin (16 mM). After 48 h, cells were stained with Annexin V and PI in Annexin binding buffer (Thermos Fisher, Grand Island, NY). Following staining, 10,000 events were collected on a FACScan (Becton Dickinson). Data were analyzed using the FCS express software (De Novo Software, Los Angeles, CA), and differences in apoptosis induction were compared using paired t tests in the SPSS 14.0 software (SPSS, Inc.).
RSCL and RRCL were exposed to DMSO or metformin (16 mM) for 48 h. Subsequently, cells were re-suspended in 0.5 ml of PBS containing 5 μmol/l of dihydrorhodamine 123 (Invitrogen) and incubated at 37 °C for 30 min in the dark. ROS was determined by flow cytometry analysis. To determine changes in the mitochondrial potential, lymphoma cell lines were exposed to metformin (16 mM) for 48 h, and 1 × 10⁶ cells were incubated in DiOC6 (Thermofisher) at 37 °C for 30 min. The dose of DiOC6 used (20 nM) is within the ranges suggested by standard protocols. Scientists had doses ranging between 10 and 20 nM [30 (link), 31 (link)]. We used FCCP treatment as a positive control. Cells were then washed and re-suspended in PBS and data collected and analyzed via flow cytometry.