Safranin: Difference between revisions

Revision as of 09:15, 10 September 2018

high-resolution terminology - matching measurements at high-resolution

Safranin

Description

Safranin is one of the most established dyes for measuring mitochondrial membrane potential by fluorometry. It is an extrinsic fluorophore with an excitation wavelength of 495 nm and emission wavelength of 587 nm. Safranin is a potent inhibitor of N-linked respiration and of the phosphorylation system.

Synonyms: Safranin O, Safranin Y, Saranin T, Gossypimine, Cotton Red, Basic Red2

Abbreviation: Saf

Reference: Kauppinen 1984 Am J Physiol, Krumschnabel 2014 Methods Enzymol, MiPNet20.13 Safranin mt-membranepotential

Template:Keywords Membrane potential

MitoPedia O2k and high-resolution respirometry: O2k-Open Support

Application in HRFR

Safranin O (C₂₀H₁₉ClN₄)

Sigma S2255, 25 or 100 mg, store at -20°C; FW = 350.84

Caution: Light sensitive (store solution in a dark vial)!

Preparation of 1 mM stock solution

Weight 3.508 mg of Safranin and dissolve in distilled water to reach 10 mL final volume;
Divide into 0.5 mL portions (use dark vials);
Store at 4 °C.

At a concentration of 2 µM, safranin can be applied for simultaneous monitoring of oxygen consumption and mtMP of mt-preparations in a CII-linked substrate state.^[1]

O2k-Procedures: O2k-Fluorometry: HRFR and simultaneous determination of mt-membrane potential with safranin. »MiPNet20.13«

Signal and output

Signal: The O2k-Fluo LED2-Module is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.
Output: The focus of the output with Safranin is on Type C: Force, mt-membrane potential.

Safranin chemical background

Several substances typically used in SUIT protocols may influence the fluorescence signal of safranin when injected into the O2k-Chamber (for instance coloured substances such as cytochrome c). These chemicals should be tested for their effect in a background run without biological sample, and the necessary corrections be applied.

Substances with an effect on the fluorescence signal of safranin

ADP (D)
Cytochrome c (c)
Succinate (S)
Rotenone (Rot)
Ascorbate (As)
TMPD (Tm)

Substances without an effect on the fluorescence signal of safranin

Pyruvate (P)
Malate (M)
Glutamate (G)
Digitonin (Dig)
Oligomycin (Omy)
CCCP (U)
FCCP (U)
Malonic acid (Mna)
Antimycin A (Ama)
DMSO
Ethanol (EtOH)
H₂O₂
H₂O

HRFR / Safranin and TMRM

The method is easy to use, since the Fluorescence-Sensor is not in direct contact with the sample. Transformation of the fluorescence signal of safranin or TMRM to mtMP [mV] requires a specific method for each preparation and protein content, which has to be kept constant in an experimental series ^[2].

» More details: »Discussion«.

References

↑ Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. »Bioblast link«
↑ Sumbalova Z, Gnaiger E (2015) High-resolution measurement of mitochondrial membrane potential and respiration – comparison of potentiometric and fluorometric methods. Abstract MiP2015. »Bioblast link«

MitoPedia methods: Fluorometry

[1] Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. »Bioblast link«

[2] Sumbalova Z, Gnaiger E (2015) High-resolution measurement of mitochondrial membrane potential and respiration – comparison of potentiometric and fluorometric methods. Abstract MiP2015. »Bioblast link«

[1]

[2]

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |abbr=Saf
-|description='''Safranin''' is one of the most established dyes for measuring mitochondrial membrane potential by [[fluorometry]]. It is an [[extrinsic fluorophores |extrinsic fluorophore]] with an excitation wavelength of 495 nm and emission wavelength of 587 nm. Safranin is a potent inhibitor of Complex I-linked respiration and of the phosphorylation system.
+|description='''Safranin''' is one of the most established dyes for measuring mitochondrial membrane potential by [[fluorometry]]. It is an [[extrinsic fluorophores |extrinsic fluorophore]] with an excitation wavelength of 495 nm and emission wavelength of 587 nm. Safranin is a potent inhibitor of [[NADH Electron transfer-pathway state |N-linked respiration]] and of the [[phosphorylation system]].
+''Synonyms:'' Safranin O, Safranin Y, Saranin T, Gossypimine, Cotton Red, Basic Red2
 |info=[[Kauppinen 1984 Am J Physiol]], [[Krumschnabel 2014 Methods Enzymol]], [[MiPNet20.13 Safranin mt-membranepotential]]
 }}
-{{MitoPedia methods
+<br />
-|mitopedia method=Fluorometry
-}}
-== Synonyms ==
-:::: Safranin O, Safranin Y, Saranin T, Gossypimine, Cotton Red, Basic Red2
+{{Keywords Membrane potential}}
-== Signal and output ==
+__TOC__
-:::# Signal: The [[O2k-Fluo LED2-Module]] is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.
+{{Template:Technical support integrated}}
-:::# Output: The focus of the output with Safranin is on '''[[O2k signals and output| Type C]]''': Force, mt-membrane potential.
 == Application in [[HRFR]] ==
-::: '''Safranin O''' (C<sub>20</sub>H<sub>19</sub>ClN<sub>4</sub>)
+:::: '''Safranin O''' (C<sub>20</sub>H<sub>19</sub>ClN<sub>4</sub>)
-::: Sigma S2255, 25 or 100 mg, store at -20°C; FW =  350.84
+:::: Sigma S2255, 25 or 100 mg, store at -20°C; FW =  350.84
+:::: <span style="color:#8B008B"> '''Caution:''' Light sensitive (store solution in a dark vial)!</span>
-<span style="color:#8B008B"> '''Caution:''' Light sensitive (store solution in a dark vial)!</span>
+::: '''Preparation of 1 mM stock solution'''
-:::'''Preparation of 1 mM solution - use for experiment''':
 ::::# Weight 3.508 mg of Safranin and dissolve in distilled water to reach 10 mL final volume;
 ::::# Divide into 0.5 mL portions (use dark vials);
 ::::# Store at 4 °C.
+:::: At a concentration of 2 µM, safranin can be applied for simultaneous monitoring of oxygen consumption and mtMP of mt-preparations in a CII-linked substrate state.<ref> Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. [[Krumschnabel 2014 Methods Enzymol |»Bioblast link«]]</ref>
+:::: [[Image:O2k-Protocols.jpg|40px|link=O2k-Procedures|O2k-Procedures]] '''O2k-Procedures''': O2k-Fluorometry: HRFR and simultaneous determination of mt-membrane potential with safranin. [[MiPNet20.13 Safranin mt-membranepotential| »MiPNet20.13«]]
-:::: At a concentration of 2 µM, safranin can be applied for simultaneous monitoring of oxygen consumption and mtMP of mt-preparations in a CII-linked substrate state.<ref> Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. [[Krumschnabel 2014 Methods Enzymol |»Bioblast link«]]</ref>
+== Signal and output ==
+:::# Signal: The [[O2k-Fluo LED2-Module]] is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.
+:::# Output: The focus of the output with Safranin is on '''[[O2k signals and output| Type C]]''': Force, mt-membrane potential.
+== Safranin chemical background ==
+:::: Several substances typically used in SUIT protocols may influence the fluorescence signal of safranin when injected into the O2k-Chamber (for instance coloured substances such as cytochrome ''c''). These chemicals should be tested for their effect in a background run without biological sample, and the necessary corrections be applied.
+=== Substances with an effect on the fluorescence signal of safranin ===
+::::* [[ADP]] (D)
+::::* [[Cytochrome c |Cytochrome ''c'']] (c)
+::::* [[Succinate]] (S)
+::::* [[Rotenone]] (Rot)
+::::* [[Ascorbate]] (As)
+::::* [[TMPD]] (Tm)
+=== Substances without an effect on the fluorescence signal of safranin ===
+::::* [[Pyruvate]] (P)
+::::* [[Malate]] (M)
+::::* [[Glutamate]] (G)
+::::* [[Digitonin]] (Dig)
+::::* [[Oligomycin]] (Omy)
+::::* [[CCCP]] (U)
+::::* [[FCCP]] (U)
+::::* [[Malonic acid]] (Mna)
+::::* [[Antimycin A]] (Ama)
+::::* [[DMSO]]
+::::* [[Ethanol]] (EtOH)
+::::* [[H2O2 |H<sub>2</sub>O<sub>2</sub>]]
+::::* [[H2O |H<sub>2</sub>O]]
+== HRFR / Safranin and TMRM ==
-:::: [[Image:O2k-Protocols.jpg|40px|link=O2k-Protocols|O2k-Protocols]] '''O2k-Protocol''': O2k-Fluorometry: HRR and simultaneous determination of mt-membrane potential with safranin. [[MiPNet20.13 Safranin mt-membranepotential| »MiPNet20.13«]]
+:::: The method is easy to use, since the [[Fluorescence-Sensor]] is not in direct contact with the sample. Transformation of the fluorescence signal of safranin or [[TMRM]] to mtMP [mV] requires a specific method for each preparation and protein content, which has to be kept constant in an experimental series <ref> Sumbalova Z, Gnaiger E (2015) High-resolution measurement of mitochondrial membrane potential and respiration – comparison of potentiometric and fluorometric methods. Abstract MiP2015. [[Sumbalova_2015_Abstract_MiP2015|»Bioblast link«]] </ref>.
+::::» More details: [[Talk:Safranin |»Discussion«]].
 == References ==
-::: <references/>
+<references/>
+{{MitoPedia methods
+|mitopedia method=Fluorometry
+}}