EVALUATION OF THE MITOCHONDRIAL STATE:

MITOCHONDRIAL TRANSMEMBRANE POTENTIAl ΔΨ

TMRE – Tetramethylrhodamine ethyl ester
The probe has lypophillic monocationic structure with delocalized charge which makes
it permeable to the mitochondrial membrane for its translocation into mitochondrial
matrix according to ΔΨ on the inner mitochondrial membrane (Plotnikov et al., 2008)
Label active mitochondria. TMRE is a cell permeant, positively-charged, red-orange
dye that readily accumulates in active mitochondria due to their relative negative
charge. Depolarized or inactive mitochondria have decreased membrane potential and
fail to sequester TMRE.
Adenosine triphosphate (ATP) is the main source of energy for metabolism.
Mitochondria provide the majority of this ATP by a process known as oxidative
phosphorylation. This process involves active transfer of positively charged protons
across the mitochondrial inner membrane resulting in a net internal negative charge,
known as the mitochondrial transmembrane potential (ΔΨm). The proton gradient is
then used by ATP synthase to produce ATP by fusing adenosine diphosphate and free
phosphate. The net negative charge across a healthy mitochondrion is maintained at
approximately -180 mV, which can be detected by staining cells with positively charged
dyes such as tetramethylrhodamine ethyl ester (TMRE). TMRE emits a red fluorescence
that can be detected by flow cytometry or fluorescence microscopy and the level of
TMRE fluorescence in stained cells can be used to determine whether mitochondria in a
cell have high or low ΔΨm. Cytochrome c is essential for producing ΔΨm because it
promotes the pumping the protons into the mitochondrial intermembrane space as it
shuttles electrons from Complex III to Complex IV along the electron transport chain.
Cytochrome c is released from the mitochondrial intermembrane space into the cytosol
during apoptosis. This impairs its ability to shuttle electrons between Complex III and
Complex IV and results in rapid dissipation of ΔΨm. Loss of ΔΨm is therefore closely
associated with cytochrome c release during apoptosis and is often used as a surrogate
marker for cytochrome c release in cells (Crowley et al., 2016)

MARCAÇÃO TMRE – PLACA PRETA 96 POÇOS

 Plaquear em placa preta de 96 poços (2,5 x105 cels/poço) (100 uL/poço)
 Incubar 24h
 Realizar protocolo de tratamento e irradiação
 Incubar por 24h
 Lavar 2x com PBS

 No controle positivo add CCCP (100 uM)  Incubar por 30 min  lavar 2x PBS
CCCP- Carbonyl Cyanide m-chloro phenul hydrazone: Inibe fosforilação oxidativa
Solução estoque: 25.000 uM (25mg CCCP + 1000 uL DMSO)
Para obter 100 uM: add 2 uL CCCP da sol. Estoque + 500 uL PBS
  Add 100 uL/poço de TMRE (0,025 uM)
Solução estoque: 1000 ug/mL ou 1942 uM em DMSO
Para obter concentração de 2,5 uM: 1,3 uL da sol. Estoque + 998,7 uL PBS
C1V1=C2V2  1942.V1=2,5.1000  V1= 1,3 + 998,7
Para obter concentração de 0,025 uM:
C1V1=C2V2  2,5.V1=0,025.30000  V1= 300 + 29700 PBS

 Incubar no escuro por 30 min a 37 ○C.

 Lavar 2x PBS
 Ler: Victor exc. 540 nm, em. 595 nm (potência da lâmpada: 20000)

 Após dosar proteína por Bradford:

Add por cima de cada poço 10 uL de TritonX-100 à 2,2% diluído em PBS (no
poço a concentração será de 0,2%)
Em placa de 96 poços transparente add 5 uL do lisado + 100 uL bradford à 25%
(diluído em água destilada).

MARCAÇÃO TMRE – MICROSCOPIA FLUORESCENCIA

 Plaquear em placa 24 poços (2,0 x105 cels/poço) (500 uL/poço)
 Incubar 24h
 Realizar protocolo de tratamento e irradiação
 Incubar por 24h
 Lavar 2x com PBS

 No controle positivo add CCCP (100 uM)  Incubar por 30 min  lavar 2x PBS
CCCP- Carbonyl Cyanide m-chloro phenul hydrazone: Inibe fosforilação oxidativa
Solução estoque: 25.000 uM (25mg CCCP + 1000 uL DMSO)
Para obter 100 uM: add 2 uL CCCP da sol. Estoque + 500 uL PBS

 Add 5 uL TMRE (2,5 uM) + 500 uL PBS (concentração no poço: 0,025 uM)
Solução estoque: 1000 ug/mL ou 1942 uM em DMSO
Para obter concentração de 2,5 uM: 1,3 uL da sol. estoque + 998,7 uL PBS

 Incubar no escuro por 30 min a 37 C.

 Lavar 2x PBS
 Visualizar no microscópio de fluorescência. Filtro 2 - Vermelho
(em lamínula em glicerol 20% - diluído em água)