Cm h2dcfda

Eight-day-old seedlings of the wild type, rres1–1, and rres1–2 were incubated in liquid MS with or without 150 mM NaCl for 12 h before the seedlings were stained. For 3′, 3′-diaminobenzidine (DAB) staining, the seedlings were immersed in 1.0 mg/mL DAB (Sigma-Aldrich) dissolved in 50 mM Tris-HCl (pH 5.0) for 10 h and then washed three times with water. The roots were then photographed using a Carl Zeiss HBO100 microscope. For chloromethyl derivative of 2′,7′-dichlorodihydrofluorescin diacetate (CM-H₂DCFDA) staining, seedlings were incubated in a buffer containing 10 μM CM-H₂DCFDA (Sigma-Aldrich) at 37 °C in darkness for 30 min and then washed with distilled H₂O to remove excess CM-H₂DCFDA. The roots were then photographed using a Carl Zeiss HBO100 microscope. ROS levels were quantified based on the intensity of fluorescence by using ImageJ software (NIH, http://rsb.info.nih.gov/ij/).

Immortalized murine alveolar macrophages, derived from a BALB/C strain mouse, (MH-S, CRL-2019, American Type Culture Collection, Manassas, VA) were cultured in a six-well plate to confluency in complete medium (RPMI 1640, 10% heat-inactivated fetal bovine serum, penicillin-streptomycin, 50 μM 2-meraptoethanol, 37 °C, 5% CO2) and treated with ferric ammonium citrate [C6H8FeNO7 dissolved in water] (FAC, 10μM, 100μM and 250μM) for 24, 72, 120 hours. Macrophage iron accumulation was assessed using the Prussian Blue Stain and staining intensity quantified by the Golde score as previously described [17 (link)]. ROS generation was determined by CM-H2DCFDA fluorimetry (3 X 10⁴ cells/well on a 96-well microplate, 37 °C, 30 min, 10 μM CM-H2DCFDA, Sigma C6827) as previously described [17 (link)] using a fluorescence plate reader (SpectraMax M3 ROM v3.0.22) at EX/Em = 485/535 nm in end point mode. For iron chelator assay, MH-S cells were treated with 30 μM of FAC for one day. The cells were washed with PBS and incubated with 100 mM of DFO at 37°C for one hour. The DFO-treated cells were incubated with 5 mM of CM-H2DCFDA at 37°C for 30 min, prior to reading with fluorescence plate reader.
Macrophage activation phenotype was assessed by quantitative PCR (qPCR) analysis of the expression of M1, M2, and MTPP genes Ccl4, Cxcl2, Cxcl10, Il1rn, and Cxcl7.

The cell-permeable oxidation-sensitive probe, CM-H₂DCFDA was used to estimate ROS generation in the primary or secondary cells as described previously [33 (link)]. Briefly, cells were harvested by centrifugation, washed three times with ice-cold PBS, re-suspended in PBS and incubated with 5 μM CM-H₂DCFDA (Sigma) for 20 min at 37 °C. After incubation, cells were again washed and lysed in PBS with 1% Tween 20. ROS level was determined at the ratio of dichlorofluorescein excitation at 480 nm to emission at 530 nm. The CM-H₂DCFDA assay is utilized as a general oxidative stress indicator and not as a detector of a specific oxidant due to known limitations of the probe [34 (link)].