Measuring ROS of Blood Cells by Flow Cytometry

Measuring ROS of blood cells by flow cytometry

Peripheral lizard blood was collected with a glass capillary tube from a vessel in the corner of the mouth, vena angularis, after being punctured with a syringe. Freshly obtained blood was diluted immediately with 9 volumes of phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, pH 7.4) and stored on ice prior to analyses, which were completed within 4 h of sampling. Prior to staining, diluted blood was diluted a further 50 fold with PBS and then centrifuged (300 g for 5 min) to pellet cells; each cell pellet corresponded to10 ml of whole blood. Cells were resuspended in 100 ml of PBS containing one of the following: no additions (unstained control), 0.1 mM dihydrorhodamine 123 (DHR; Molecular Probes, Invitrogen, USA), or 5 mM MitoSOX Red (MR; Molecular Probes, Invitrogen, USA). DHR and MR added from stock solutions in dimethylsulfoxide (DMSO); the final concentration of DMSO was 0.2% (v/v) or less. Cells were subsequently incubated at 37 oC for 30 min, then washed with PBS by centrifugation as described above and held on ice until analysed by flow cytometry; 50,000 events were acquired for all samples. Flow cytometry was performed using a Becton Dickinson LSR II, with excitation at 488 nm and emitted fluorescence collected using band pass filters of 515 ± 10 nm (DHR) and 575 ± 13 nm (MR). Data was acquired and analysed using FACSDiva v4.0.1 (Becton Dickinson, Sydney, Australia) and FloJo v8.8.7 (Treestar Inc., USA) software, respectively. On the basis of forward angle laser scatter and side angle laser scatter, a number of blood cell populations were discerned; the results obtained were similar for all these populations. For each sample, the mean fluorescence for all 50,000 cells acquired was determined using FloJo software and used to compare between samples.

Both DHR and MR are cell permeable and accumulate in mitochondria within live cells. DHR becomes fluorescent when oxidised by H2O2 or peroxynitrite [1,2]. Thus DHR was used as a probe for a variety of forms of ROS. MR becomes fluorescent when it is specifically oxidised by superoxide (SO). We have shown that carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma, Sydney, Australia), a proton ionophore and mitochondrial uncoupler which increases the rate of operation of the mitochondrialelectron transport chain [3], also increases SO levels in lizard cells[4]. This shows thatmetabolism contributes to SO production.

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[4] Olsson, M., Wilson, M., Uller, T., Mott, B., Isaksson, C., Healey, M., and Wanger, T.

2008 Free radicals run in lizard families. Biol. Lett. 4, 186-188.