For ospA and ospC, the first round of amplification was carried out using a touchdown protocol; after an initial denaturation step of 95°C for 5 min, 2 cycles of 95°C for 1 min, 64°C for 1 min, 72°C for 1 min were run, followed by decreasing the annealing temperature by 1°C per 2 cycles until reaching an annealing temperature of 55°C, used for the next 17 cycles.
Gradients of B in 30 min were run at flow rates of 0.8 mL/min and 4 mL/min for analytical and preparatory procedures, respectively.
Gradients of 125 min were run at a flow rate of 150 nL/min (A-solvent: 0.2 % formic acid in water and B: 100 % acetonitrile).
Initially, an exploratory gradient from 5 to 100% (B) in 60 min was run as suggested by Snyder et al. (1997) [ 18].
Cells treated with 50 μ M of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) at 37°C for 15 min were run in parallel as a control for the collapse of mitochondrial transmembrane potential.
The reaction (95°C for 15 s and 60°C for 1 min) was run using the ABI PRISM 7500 Real-Time PCR System (Applied Biosystems) with relative expression analyzed by the 2−ΔΔCt method.
Subsequently, an elution gradient of 5-35% acetonitrile (0.1% formic acid) over 70 min was run with an in-house packed analytical column (75 μm × 15 cm, C18, 3 μm 150 Å) with a spray tip.
After initial steps at 50°C for 2 min (UNG activity) and at 95°C for 10 min (activation of the Ampli Taq Gold polymerase), a two-step program of 95°C for 15 s and 62°C for 1 min was run for 40 cycles.
Each PCR cycle included incubations at 94°C for 30 s, at 55°C for 1 min, and at 72°C for 5 min. One additional cycle at 72°C for 10 min was run after the last cycle to allow trimming of incomplete polymerizations.
Amplification was performed using Go Tag DNA Polymerase (Promega) with the following thermal conditions: 95°C for 2 min followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec and as a last step, final extension at 72°C for 10 min was run.
