Dihydrorhodamine 123 dhr

MCF-7 and MDA-MB
231 cells (1 × 10⁴ per well) were seeded into a 96
well culture plate in complete growth medium. After 24 h, the cells
were treated with the complexes or the ligand in the same medium for
3 h. After a wash with PBS, 1.5 μM of the probe dihydrorhodamine
123 (DHR) (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS-glucose
(10 mM) was added to the cells and the fluorescence increase was monitored
at λ_ex = 485 nm and λ_em = 527 nm
using a plate reader (Tecan Infinite M200 PRO, Männedorf, CH).

Cells (300,000) were stained with anti CD11b-APC (clone M1/70, Biolegend) before being incubated for 15 min at 37 °C in 1 mL of PBS-gg (0.05% gelatine, 0.09% D-glucose) containing 2.9mM of dihydrorhodamine 123 (DHR; Sigma - Aldrich) and 150U/mL of catalase (Sigma-Aldrich). The samples were then divided into two equal portions (500 mL each) with one receiving 1 mg/mL of PMA (Sigma-Aldrich) and were incubated at 37 °C for further 15 min before being analysed on the LSRII flow cytometer.

The production of reactive oxygen species (ROS) was assessed using dihydrorhodamine 123 (DHR; Sigma-Aldrich) as described previously^{60 (link)}. Cells were grown in YP galactose medium until mid exponential phase, a sample was collected and 100 mM glucose was added after which a new sample was taken. 5 µg/mL DHR was added to the cells and incubated for 2 h. ROS production was visualized using fluorescence microscopy (Axioplan 2 imaging, Zeiss).

Ficoll-Paque was obtained from GE Healthcare (Uppsala, Sweden). Human Serum Albumin (HSA) was from Sanquin (Amsterdam, the Netherlands). HEPES-buffered RPMI 1640 was from Invitrogen (Carlsbad, CA, USA). Dihydrorhodamine 123 (DHR) was purchased from Sigma Aldrich (St. Louis, MO, USA) and dissolved in DMSO at a concentration of 3,33 mg/ml and stored at −20 °C. Platelet Activating Factor (PAF) and fMLF were purchased from Sigma Chemical Co (St. Louis, MO, USA). The VIM12 (CD11b, Mac-1, IgG1) was obtained from Caltag Invitrogen (Carlsbad, CA, USA). For flow cytometry staining we used the antibodies CD18 (clone L130); CD15 (clone MMA); CD32 (clone FLI8.26), CD35 (clone E11), CD44 (clone 515), CD63 (clone H5C6) and CD16 (clone 3G8) obtained from BD Pharmingen (San Diego, CA, USA). CD11b (clone 2LPM19c) was from DAKO (Copenhagen, Denmark), CD66b (clone 80H3) was from Cytognos (Salamanca, Spain), CXCR1 (clone 42705), CXCR2 (clone 48311) and CD45 (clone 2D1) were from R&D systems (Europe, UK) and CD64 (clone 10.1) was from AbD Serotec (Oxford, UK). CD29 (clone N29) was purchased from Millipore. All other chemicals were reagent grade.

Isolated neutrophils from healthy donors (2 × 10⁴ cells) were cultured in the presence of HBSS medium containing 25% of cf-MPE-LAC, 25% cf-PE-HF or 2% BSA as a control [22 (link)]. Briefly, cells were first incubated in the presence of dihydrorhodamine 123 (DHR; Sigma-Aldrich) for 10 min and then stimulated with PMA (325 nM; Sigma Aldrich) for 30 min or remained unstimulated in a humidified incubator (37 °C). ROS production was measured by oxidation of DHR to rhodamine by flow cytometry. Data were expressed as oxidative burst indexes (MFI of DHR under pleural fluid culture conditions divided by the respective nonpleural fluid culture control).

Dulbecco's-modified Eagle medium (DMEM), RPMI-1640 medium, fetal calf serum (FCS), penicillin/streptomycin, NAD⁺, NADH, dihydrorhodamine 123 (DHR), HBSS, MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and PES (phenazine ethosulfate) were from Sigma-Aldrich (St. Louis, MO, USA). Glutamax and DPBS were from Life Technologies (Waltham, MA, USA). XF Cell Mitostress kit was from Seahorse Bioscience (North Billerica, MA, USA). Horse serum (HS) was from Amimed, Bioconcept (Allschwil, Switzerland).

Analysis of superoxide and peroxynitrite production by total leukocytes was performed as previously described [17 (link)]. Following preincubation of blood samples with compstatin (1 mM) for 10 min at room temperature and stimulation with PBS, LPS, or P-MAPA (described in Section 2.4), aliquots of blood (50 μL) were incubated with 1 μL of dihydroethidium (DHE) or dihydrorhodamine 123 (DHR) (5 μM) (both from Sigma) at 5% CO₂ for 1 h at 37°C, for DHE, or 30°C, for DHR. Following incubation, erythrocytes were lysed with BD FACS Lysing Solution, according to the manufacturer's instructions, and samples were centrifuged (720 g, 4°C, 10 min). Supernatants were discarded and cells were washed in FACS buffer, fixed with 0.5% paraformaldehyde and analyzed by flow cytometry.