User talk:Wiethuechter Anita

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2011-10-06: Cell experiment - oxidative stress

Protocol: 2011-10-06

Oxygraphs

  • Medium: MiRO5 for air calibration
  • T: 37°C, Gain 1

1) remove EtOH from the chamber and wash 3 times with water 2) add Medium and make an air calibration (R1) 3) remove medium after air calibration and add "warm" cell suspension (at least 2.2 ml; more if subsamples are needed) 4) stirr with open chamber for 5 min to allow Temperature adjustment 5) close the stopper and start a new DatLab file 6) Layout 05: to check the flux (~ 20 pmol s-1 cm-3 is optimal) 7) Layout06_specific flux per unit samples during the experiment

Time Regime

  • ROUTINE: for 15 min
  • add H202 or H20 (4 µl): for 20 min
  • add catalase (5 µl): "open" the stopper for air calibration; 10 min
  • close the stopper: for approx. 5 min (until stable signal)
  • start the FCCP titration until optimal conc. is reached
  • add Rotenone: 10 min
  • add Mna, Ama: 10-15 min for ROX


Experimental setup:

Titrations (µl = final conc. in the 2 ml chamber): H202/H20 (4 µl = 500 µM) + Catalase (5 µl = 280 u/ml) + Omy (1 µl = 2µg/ml) + F (1µl steps = 0.5 µM steps) + Rot (2 µl = 0.1 µM) + Mna (5 µl = 5 mM) + Ama (1 µl = 2.5 µM)

A => acute (H20) + Cat + Omy + FCCP + Rot + Mna + Ama
B => stress(H202) + Cat + Omy + FCCP + Rot + Mna + Ama
C => acute (H20) + Cat + Omy + FCCP + Rot + Mna + Ama
D => stress(H202) + Cat + Omy + FCCP + Rot + Mna + Ama
E => acute (H20) + Cat + EtOH + FCCP + Rot + Mna + Ama
F => stress(H202) + Cat + EtOH + FCCP + Rot + Mna + Ama
G => acute (H20) + Cat + EtOH + FCCP + Rot + Mna + Ama
H => stress(H202) + Cat + EtOH + FCCP + Rot + Mna + Ama

Cells:

MEFS: mouse embryonic fibroblasts in cell culture medium
cell concentration: