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. Author manuscript; available in PMC: 2009 Jul 16.
Published in final edited form as: Nat Protoc. 2007;2(3):512–522. doi: 10.1038/nprot.2007.5

Figure 4.

Figure 4

Immuno-spin trapping of DNA radicals in macrophages. (a) We loaded RAW 264.7 macrophages (70% confluence) with 100 µM of either ferric citrate (yellow bars) or cupric chloride (blue bars) in complete medium (DMEM plus 10% (vol/vol) FCS) for 18 h in an incubator. After washing with HBSS− plus 1 mM DTPA (HBSS−/DTPA), we harvested and washed the cells with pre-warmed (37 °C) HBSS−/DTPA. We counted the cells and ensured that their viability was >90%. We then divided the cells (106 cells per ml) in clean, clear Eppendorf tubes in 1 ml HBSS−/DTPA and added 100 mM DMPO from a 10-M stock DMPO solution. As a control, we ran experiments in which we added, at the same time as DMPO, the cell-permeable iron and copper chelators 1 mM DP and 100 µM sodium diethyldithiocarbamate (DETC), respectively. We added different concentrations of tert-BOOH (as 100 X stock in HBSS−; <0.1% DMSO) and incubated the tubes at 37 °C for 1 h. Finally, we washed the cells with HBSS−/DTPA, counted them, and determined their viability (green bars) using hemocytometer counting and Trypan blue. We washed the cells twice with HBSS−/DTPA and pulled them down for DNA extraction. We loaded purified DNA samples onto two separate microtiter plates. After coating, one of the plates was washed with phosphate buffer and used to determine bound DNA using a CyQUANT cell proliferation assay kit (Molecular Probes, Invitrogen, cat. no. C7026) that uses λ DNA as standard. We washed the other plate with washing buffer to determine nitrone adducts using HRP as the detection system (see PROCEDURE). The results are the mean values of DNA nitrone adducts per mg DNA ± s.e.m. from three experiments performed in triplicate (n = 9). *P < 0.05 with respect to cells without added tert-BOOH. r.l.u., relative light units. (b) We analyzed the samples from (a) using the immuno-slot blot procedure (option II). Nitrone adducts from cells loaded with copper and treated with tert-BOOH, and immunocomplexes detected using HRP-conjugated secondary antibodies, are shown. 100# indicates macrophages pre-treated with DETC before treatment with tert-BOOH.