User talk:Wiethuechter Anita
From Bioblast
2011-10-06: Cell experiment - oxidative stress
Protocol: 2011-10-06
Oxygraphs
- Medium: MiRO5 for air calibration
- T: 37Β°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 Β΅l): for 20 min
- add catalase (5 Β΅l): "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 (Β΅l = final conc. in the 2 ml chamber): H202/H20 (4 Β΅l = 500 Β΅M) + Catalase (5 Β΅l = 280 u/ml) + Omy (1 Β΅l = 2Β΅g/ml) + F (1Β΅l steps = 0.5 Β΅M steps) + Rot (2 Β΅l = 0.1 Β΅M) + Mna (5 Β΅l = 5 mM) + Ama (1 Β΅l = 2.5 Β΅M)
- 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: