Measuring respiration in cultured cells

HSM Buffer: 20mM HEPES (pH7.1 with KOH), 250mM Sucrose, 10mM MgCl₂ (store it at -20^o)

Substrate and Inhibitor stocks:

Malate 1M

Glutamate 1M

Succinate 0.5M

Glycerol-3-Phospahte 0.5M

Ascorbate 1M

TMPD 20mM (made fresh in HSM)

Rotenone 2Mm (in EtOH)

Malonate 0.5M

Antimycin 4Mm (in EtOH)

KCN 0.1M (in HSM and Ph 8.0)

*pH of all Substrate solutions (except TMPD) should be adjusted to 7.0 and stored at -20^o. Inhibitors, excluding KCN, can be stored at -20^o. KCN can be stored at -80^o

Digitonin Permiabilization of Cells

· 2 x 10⁷ cells (freshly harvested) suspended in 1ml HSM buffer on ice.

· 1 m l of 10% Digitonin (in DMSO) added to the cell suspension and left on ice for 5 min.

· 5ml HSM added, mixed and centrifuged at R/T for 5 minutes.

· The cell pellet washed with 5ml of HSM and centrifuged at R/T for 5 minutes.

· The pellet is suspended at 2mg/ml protein concentration and frozen as 0.5ml aliquots (i.e., 1mg protein)

Setting-up the Electrode

1. Prepare the Electrode disc with a fresh membrane.

· Read either the printed manual or the online help files from Hansatech. They are very informative.

· Wash the electrode chamber with new membrane at least three times with Di H₂O.

· If the silver anode is has black/brown electrochemical deposit, clean it by applying No.1 Coarse polishing paste with a cotton bud.

2. Calibrating the electrode.

· Place 1-1.5ml of a i r-saturated de-ionized water at 37°C into the electrode reaction chamber, add the magnetic follower and stir it for at least 15 min.

· Flush the O₂, to establish the zero line. This is done by adding either Sodium dithionate or Sodium Sulfate to the water in the chamber to reduce all the dissolved oxygen.

· The signal recorded on the trace should fall as the oxygen concentration in the electrode disc falls to zero. Ideally electrical zero would coincide with zero oxygen. In practice there is always some minor current flow (residual signal) that occurs in the absence of oxygen.

· When all of the calibration data has been collected the software calculates the difference between the two measured signals, the air-line and zero oxygen line, from the electrode. This is end of calibration.

· Wash the electrode chamber thoroughly with Di water at least 10 times.

· Put 1ml of air saturated Di water at 37°C in the chamber and stir it till there are no air bubbles on the electrode surface.

Measuring the O₂ Consumption

The frozen permiabilized cell suspension is thawed instantly, by either incubating it in a 37^o water bath or worming up on the hands, and transferred into the empty calibrated electrode chamber. The O₂ consumption rates are measured by adding specific substrates and inhibitors to the cell suspension, over the electrode, at constant stirring and is equilibrated at 37^o.

Complex I: When the reading is stable after adding the thawed cells (in about two minutes) add 5 m l of Glutamate-Malate (1:1 mixture of1M stock solution makes the working solution) 1M Na-Glutamate (effective 5mM). Once the signal reaches a platue (i.e., the consumption rate is about -2), normally in about 2min, 5 m l of 10 m M Rotenone is added to inhibit the enzyme.

Complex II: Once the maximal inhibition of complex I by rotenone is reached 5ml of 0.5M Succinate (effective 5mM) is added to the reaction mixture. After appropriate time, normally with-in a minute, 5ml of 0.5M Malonate (5mM effective) is added to inhibit the activity.

Complex III: 5 m l of 0.5M G3P (effective 5mM) and 5 m l of 2 m M Antimycin (20nM final) are added as substrate and inhibitor respectively after the inhibition of complexes I and II.

Complex IV: Add 10 m l of TMPD/Ascorbate mixture (1:1 20mM TMPD and 1M Na-Ascorbate) as substrates (0.2mM TMPD and 10mM Ascorbate final) and 5 m l of 0.1M KCN as an inhibitor (0.1mM final) once the upstream complexes were inhibited.

Cleaning the Electrode Chamber

The inhibitors rotenone and antimycin are very powerful and tend to stick to the glass chamber, membrane and syringes. Their residues effect the latter measurements unless the syringes and the chamber are rinsed with EtOH and then with water. Finally, washing the chamber with 10% BSA solution (once) and with water several times (at least 6 times) after the EtOH cleaning would remove any sticking inhibitors.

Hofhaus, G., Shakeley, R. M., & Attardi, G. (1996). Methods Enzymol. 264, 476483