mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism .
they can be obtained quickly and reliably in great quantity from wild type or knock - out animals .
there are several conditions that have to be kept during isolation of mitochondria from various tissues , including the heart ( fig .
1 ) . the tissue has to be thoroughly minced , since it directly affects the yield of obtained mitochondria .
heart tissue is best to mince with small curved scissors and can be followed by use a latapie tissue grinder or , if a tissue grinder is not available , a garlic press with outlet holes diameter of around 1.0 mm diameter .
therefore the entire procedure has to be carried out in a cold room and all instruments and solutions have to be prepared and cooled in advance .
if the temperature conditions are not kept stable several properties of the preparation can be lost .
mitochondrial structure is very sensitive to freezing so overfreezing of the suspension ( for example during centrifugation ) is not permitted , especially if studies of active transport are important .
an important parameter is the time - length of the procedure ; the isolation should be performed as quickly as possible and the obtained preparation has to be used immediately .
therefore , surgical instruments , solutions and appliances have to be prepared in advance .  
straight operating scissors , sharp point   small funnel + piece of nylon filter ( prewashed )   two magnetic stirrers and two ice baths   2 small magnetic bars   spatulas for pellet scraping   latapie tissue press ( can be substituted with garlic press )   teflon - glass homogenisers 20ml and 1 - 2ml   eppendorf tubes for final mitochondrial suspension and samples for protein determination   washing buffer : 0.3 m sucrose , 10 mm hepes ( ph=7.2 ) , 0.2 mm edta ( 1000 ml )   isolation buffer : 0.3 m sucrose , 10 mm hepes ( ph=7.4 ) , 0.2 mm edta , 1 mg / ml bsa ( sigma a6003 ) ( 100ml prepared from the washing buffer ) . bovine serum albumin can be substituted by less expensive fatty acid - free analogues .
trypsin ( sigma t8003 ) solution : 2.5mg / ml in 1 mm hcl ( 0.5 ml )   trypsin inhibitor from soy bean ( sigma t9128 ) 6.5 mg/10 ml of isolation buffer the general scheme of the procedure is shown on fig .
1 . hearts should be cooled and washed in washing buffer , ventricular tissue excised , minced and homogenised during photolytic treatment with trypsin .
the suspension is centrifuged at low speed and the obtained supernatant is centrifuged again at higher speed to collect mitochondria .
depending on the planned experiments the resuspending buffer can be replaced by any other isotonic solution .
" animals ( scientific procedures ) act . " by cervical dislocation followed by decapitation .
it is necessary to extract the heart immediately after decapitation of the animal , so that there is no delay between animal termination and heart extraction .
if parallel isolation of liver mitochondria is expected the rats should be fasted overnight prior to the experiment .  
cool them in the ice - salt bath for 3 to 5 min before proceeding to the next step .
make sure not to overfreeze them and use immediately just after clouds of ice crystals start to appear .  
make small incisions and then immediately transfer the heart to the ice - cold solution in the first beaker .
remove all fat , clotted blood , auricles and fasciae and pool the ventricular tissue .  
mince the pooled tissue with small curved scissors on the petri dish on ice for around 5 min until the size of the particles is about 1 - 2 mm .   put the minced tissue into the tissue press and pass it through .
be aware that a substantial amount of the material can remain in the press so collect all the heart tissue from the walls and bottom of the press .  
wash the minced tissue on the filter 3 times with the washing buffer and discard the filtrate .  
transfer the washed tissue into 20 ml of washing buffer and place it in the ice bath on the magnetic stirrer .
prepare another magnetic stirrer , ice bath and a second 50 ml beaker .   on the 4 minute briefly homogenise the suspension portion by portion using a small loosely fitted glass - teflon homogeniser ( 10 - 15ml ) with several up and down strokes to disperse the suspension .
repeat the procedure on the 9 minute , so that you perform the homogenisation of the suspension two times in total .
after 15 min of incubation , add 10 ml of isolating buffer containing trypsin inhibitor and incubate for 1 min .  
filter the suspension again through a nylon filter and save the filtrate . collect the particulate fraction left on a filter and homogenise for 1 min in 15 - 20 ml of washing buffer .  
combine the homogenate with the filtrate and centrifuge for 15 min at 600 g .   carefully filter the resulting supernatant into a new centrifuge tube . avoid contamination by the pellet and centrifuge again for 15 min at 8500 g .   after high speed centrifugation
discard the supernatant and rinse the pellet 2 - 3 times with 1 ml of the buffer discarding the fluffy white outer rim layer .
break up the pellet with the spatula but avoid the red spot of erythrocytes at the bottom .
if the blood cells got loos , remove the red bits from the suspension before homogenisation .  
dilute the suspension with more isolationbuffer and centrifuge again for 15 min at 8500 g .  
discard the supernatant , resuspend the pellet in resuspending buffer ( an isotonic buffer of choice ) and centrifuge again .  
top part , stages 1 - 9 : tissue disruption , trypsin treatment and homogenisation ; bottom part , stages 10 - 15 : differential centrifugation .
mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism .
they can be obtained quickly and reliably in great quantity from wild type or knock - out animals .
there are several conditions that have to be kept during isolation of mitochondria from various tissues , including the heart ( fig .
1 ) . the tissue has to be thoroughly minced , since it directly affects the yield of obtained mitochondria .
heart tissue is best to mince with small curved scissors and can be followed by use a latapie tissue grinder or , if a tissue grinder is not available , a garlic press with outlet holes diameter of around 1.0 mm diameter .
therefore the entire procedure has to be carried out in a cold room and all instruments and solutions have to be prepared and cooled in advance .
if the temperature conditions are not kept stable several properties of the preparation can be lost .
mitochondrial structure is very sensitive to freezing so overfreezing of the suspension ( for example during centrifugation ) is not permitted , especially if studies of active transport are important .
an important parameter is the time - length of the procedure ; the isolation should be performed as quickly as possible and the obtained preparation has to be used immediately .
  straight operating scissors , sharp point   small funnel + piece of nylon filter ( prewashed )   two magnetic stirrers and two ice baths   2 small magnetic bars   spatulas for pellet scraping   latapie tissue press ( can be substituted with garlic press )   teflon - glass homogenisers 20ml and 1 - 2ml   eppendorf tubes for final mitochondrial suspension and samples for protein determination
  washing buffer : 0.3 m sucrose , 10 mm hepes ( ph=7.2 ) , 0.2 mm edta ( 1000 ml )   isolation buffer : 0.3 m sucrose , 10 mm hepes ( ph=7.4 ) , 0.2 mm edta , 1 mg / ml bsa ( sigma a6003 ) ( 100ml prepared from the washing buffer ) .
trypsin ( sigma t8003 ) solution : 2.5mg / ml in 1 mm hcl ( 0.5 ml )   trypsin inhibitor from soy bean ( sigma t9128 ) 6.5 mg/10 ml of isolation buffer
1 . hearts should be cooled and washed in washing buffer , ventricular tissue excised , minced and homogenised during photolytic treatment with trypsin .
the suspension is centrifuged at low speed and the obtained supernatant is centrifuged again at higher speed to collect mitochondria .
depending on the planned experiments the resuspending buffer can be replaced by any other isotonic solution .
animals were terminated in accordance with the u.k . " animals ( scientific procedures ) act . " by cervical dislocation followed by decapitation .
it is necessary to extract the heart immediately after decapitation of the animal , so that there is no delay between animal termination and heart extraction .
if parallel isolation of liver mitochondria is expected the rats should be fasted overnight prior to the experiment .  
cool them in the ice - salt bath for 3 to 5 min before proceeding to the next step .
make sure not to overfreeze them and use immediately just after clouds of ice crystals start to appear .  
make small incisions and then immediately transfer the heart to the ice - cold solution in the first beaker . squeeze the heart to remove blood and put it into the second beaker
remove all fat , clotted blood , auricles and fasciae and pool the ventricular tissue .   mince the pooled tissue with small curved scissors on the petri dish on ice for around 5 min until the size of the particles is about 1 - 2 mm .  
be aware that a substantial amount of the material can remain in the press so collect all the heart tissue from the walls and bottom of the press .  
transfer the material into the beaker with 50 ml washing buffer and stir . then filter the suspension through a nylon filter .
wash the minced tissue on the filter 3 times with the washing buffer and discard the filtrate .  
transfer the washed tissue into 20 ml of washing buffer and place it in the ice bath on the magnetic stirrer .
prepare another magnetic stirrer , ice bath and a second 50 ml beaker .   on the 4 minute briefly homogenise the suspension portion by portion using a small loosely fitted glass - teflon homogeniser ( 10 - 15ml ) with several up and down strokes to disperse the suspension .
repeat the procedure on the 9 minute , so that you perform the homogenisation of the suspension two times in total .
after 15 min of incubation , add 10 ml of isolating buffer containing trypsin inhibitor and incubate for 1 min .  
filter the suspension again through a nylon filter and save the filtrate . collect the particulate fraction left on a filter and homogenise for 1 min in 15 - 20 ml of washing buffer .  
combine the homogenate with the filtrate and centrifuge for 15 min at 600 g .   carefully filter the resulting supernatant into a new centrifuge tube .
avoid contamination by the pellet and centrifuge again for 15 min at 8500 g .  
after high speed centrifugation discard the supernatant and rinse the pellet 2 - 3 times with 1 ml of the buffer discarding the fluffy white outer rim layer .
break up the pellet with the spatula but avoid the red spot of erythrocytes at the bottom .
if the blood cells got loos , remove the red bits from the suspension before homogenisation .  
dilute the suspension with more isolationbuffer and centrifuge again for 15 min at 8500 g .  
discard the supernatant , resuspend the pellet in resuspending buffer ( an isotonic buffer of choice ) and centrifuge again .  
top part , stages 1 - 9 : tissue disruption , trypsin treatment and homogenisation ; bottom part , stages 10 - 15 : differential centrifugation .
detailed rapid protocol for isolation of intact mitochondria with a high degree of coupling is described .
obtained preparation can be used for functional or structural research on cellular bioenergetics , proteomic studies or analysis of mitochondrial dna and lipid content .
it is possible to further process the mitochondrial preparation in order to isolate submitochondrial particles , outer membrane or purify separate components of oxidative phosphorylation .
the described method is a modification of the standard protocol of isolation by jacobus and saks .
the expected yield of the prepared mitochondria is approximately 10 - 15 mg of mitochondrial protein per heart and the respiratory control ratio on malate / glutamate of such preparation varies between 8 to 12 with state iv respiration of around 0.12 mol o2xminxmg protein at 23c in the medium comprising 0.25 m sucrose , 10 mm kcl , 0.2 mm edta , 5 mm potassium phosphate ( ph 8.0 ) with 150 m adp for initiation state iii respiration ( for the exact experimental conditions and additions , see ref . ) .
special attention should be given to several technical details before the starting of the isolation procedure .
it is crucial to use clean polycarbonate or polypropylene centrifuge tubes , therefore tubes should be thoroughly washed with a clean brush and hot water , however the detergent treatment should be kept at the minimum .
also , it is better to collect all necessary glassware and instruments in the cold room a day before the isolation . during the isolation when transferring buffers from flasks to beakers ,  special attention should be given to prevent drops of ice water into the solutions .
when handling tissues it should be stressed that heart extraction should be carried out as soon as possible since even short - time delay between decapitation of the animal and cooling the tissue would result in partially uncoupled mitochondria .
rapid cooling and thorough washing of removed hearts is essential to obtain standard preparation , free from haemoglobin and erythrocyte nadase .
trypsin is used for connective proteins degradation to increase susceptibility of the tissue to shearing force during homogenisation .
prolonged exposure of the tissue to protease should be avoided since it results in mitochondria of inferior quality . after high speed centrifugations the walls of the centrifuge tubes should be wiped with tissue paper to remove any deposits of fat material accumulated on the walls . for the final resuspension it is better to use a low volume of the final buffer in order to minimise loss of mitochondrial functions during storage ( 150 to 200 l of the buffer per heart ) . for studies of the transport of divalent cations
no chelating agents must be present in the final preparation thus mitochondria should be washed for several times with egta - free medium and used within first hours after isolation .
 the function of mitochondria in generation of cellular atp in the process of oxidative phosphorylation is widely recognised . during the past decades 
 there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy . 
 these include their role in apoptosis , acting as signalling organelles , mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation . 
 our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals . 
 mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock - out animals.here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling . 
 such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics , transport of biomolecules , proteomic studies and analysis of mitochondrial dna , proteins and lipids . 
Protocol
Introduction
Materials and instruments
Instruments
Solutions (calculated per 2 rats):
Protocol
Discussion
Disclosures