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. 2019 Dec 27;3(2):e201900376. doi: 10.26508/lsa.201900376

Figure 2. Flow cytometric and electron spin resonance detection of reactive oxygen species.

Figure 2.

PBMCs isolated from patient 4 (see main text) and 3 healthy controls (HC) were stained for 15 min with 5 μM MitoSox or 10 μM DHE and analysed immediately by flow cytometry. Dotted lines in (A, B, C, D) denote HC (one representative experiment); red lines denote patient 4. (A, B) Mitochondrial oxide in monocytes (A) showed no significant difference, but (B) was increased in lymphocytes from patient 4 (***P < 0.001, two-tailed Z-test). (C, D) Oxidative stress detected by H2DCFDA was elevated in both patient monocytes (C) and lymphocytes (D). Fig 2E–H: 500,000 THP1 cells were seeded into a 24-well plate with 100 nM PMA. After 24 h, the medium was replaced and lipofectamine and siRNA complexes were added. After a further 24 h, the medium was replaced with RPMI with 2% FCS for another 24 h. (E, F, G, H) Cells were then trypsinized and either stained for 15 min with 5 μM MitoSox (E), 10 μM DHE (F), or 10 μM H2DCFDA (H) and analysed immediately by flow cytometry; or the cells were resuspended in antioxidant buffer, spin probe CMH was then added, and superoxide production was measured 10 times over 10 min via electron spin resonance spectroscopy (G). Cytoplasmic superoxide was significantly increased in MEFV knockdown (kd) cells compared with scrambled control. Mitochondrial superoxide, superoxide production, and oxidative stress levels were significantly increased in the TRAP1 kd cells. (I) THP1 cells were transfected with full-length C-terminal DDK-tagged TRAP1 in a pCMV6-Entry vector, with either wild-type TRAP1 sequence or with the same point mutation inducing the p.R128H protein change or the empty vector alone. 500,000 THP1 cells were seeded into a 24-well plate in RPMI with 10% FCS and 100 nM PMA overnight. Relevant wells were treated with 5 μM CCCP for 6 h. The cells were then stained with 5 μM MitoSox, trypsinized, and analysed immediately by flow cytometry. At baseline, MitoSox fluorescence was slightly but significantly decreased in only the WT-TRAP1 overexpressing cells compared with the vector control. Upon treatment with CCCP, mitochondrial superoxide increased in vector control and R128H mutant TRAP1, but to a significantly lesser extent in the WT-TRAP1 cells. kd, knockdown; WT, wild-type.