Mitochondria Have numerous roles in cell physiology from metabolism and energy production to program cell death to steroid hormone synthesis. Despite the importance of mitochondria in both fundamental cellular functions and disease, very little is known about the function of mitochondria in human embryonic stem cells and their differentiated progeny. In this video, we will show you how the mitochondria respiratory chain complexes of human amry stem cells can be analyzed using Ingel activity assays.
Hi, I am Ivan OV from the laboratory of Michael Tetel in the Department of Pathology and Laboratory Medicine at the University of California Los Angeles. Today we will show you a procedure for assessing mitochondrial respiratory complex function in human embryonic stem cells. These in gel activity assess this procedure involves isolation of crude mitochondrial palate from human embryonic stem cells, separation of mitochondrial complexes of the exudative phosphorylation system with higher resolution clear native poly acrylamide gel electrophoresis, and subsequent in gel activity sa.
So let's get started. The first step in this procedure is to isolate mitochondria from human embryonic stem cells. For this assay, we'll use human embryonic stem cells that have been grown on a matrigel covered plate in a feeder free condition.
In order to collect mitochondria from our human amry stem cells, we start by washing them with warm one XPDS pH 7.4, and then trypsin the cells for five minutes. You can use one mil of one x trypsin per well in a six well plate. After five minutes, you can stop the reaction by adding an equal volume of fresh trypsin inhibitor one milligram per mil in one XPBS and harvest the cells.
The collected cells are then centrifuge at 200 times G for five minutes at room temperature. After spinning, discard the supernatant and resus, suspend the pelleted cells in 0.8 to 1.7 milliliters of ice cold mitochondria isolation buffer with a freshly prepared cocktail of one x protease inhibitor. How much buffer you use depends on the size of the cell pellet you get After the pellet has been resuspended, incubate it on ice for about 10 minutes.
Following the incubation on we down the cells with a homogenizer, this might take about 40 to 50 strokes. We can check for the complete list of homogenization by staining an aliquot of the DOD cells with trian blue and looking through a light microscope. It should look something like this.
This is the first shot after 50 strokes with a downs. This is the second shot after 80 strokes with a downs. Once the cells are homogenized, centrifuge them at 20, 000 times G for 10 minutes at four degrees C.This will give us a crude mitochondrial pellet, which will also contain nuclei and larger cell fragments.
Before collection of the pellet, we have to weigh the collection tube in order to estimate the subsequent pellet weight, pour out and discard the supernatant, and then weigh the precipitated crude mitochondrial pellet. By subtracting the weight of the tube at this point, one can proceed to solubilizing the pellet, which will be shown in the next step. In order to solubilize the crude mitochondrial pellet, we start by keeping the pellet on ice and vortexing with 35 microliters of ice cold solubilization buffer.
For every 20 milligrams of pellet that we have detailed information on the buffer compounds is mentioned in the accompanying written protocol. Following vortexing and addition of solubilization buffer and before adding digit toin, we transfer the resuspended pellet into a tube appropriate for high speed centrifugation, and then we add 20 microliters of 10%weight per volume digit toin for 20 milligrams of pellet weight and mix by flicking the tube after it has been mixed thoroughly incubate the mitochondrial pellet for five to 10 minutes on ice. Doil beta D multiside or DDM or Triton X 100 can be substituted for digit toin to solubilized proteins.
The choice of detergent and its quantity may affect the formation of supra complexes or multimeric forms of mitochondrial complexes following the incubation on ice. And once the pellet has been solubilized, we'll centrifuge it at a hundred thousand times G for 15 minutes at four degrees C, we will then collect the clear supernatant. We are now ready to prepare the supernatant for running on our gradient gel for native gradient gel electrophoresis of mitochondria.
We recommend using an acrylamide gradient gel from five to 13%with a 3.5%stacking gel. To prepare the gel, we will use an acrylamide bis acrylamide mix as described in the written protocol. Acrylamide bis acrylamide solution is prepared in advance and stored at four degrees Celsius in a dark place.
Acrylamide and bis acrylamide are toxic chemicals both as a powder and in liquid form, so special attention should be paid when you work with them. We combine the Acrylamide bis acrylamide solution with an aliquot of three x gel buffer composed of 1.5 molar six amino hexa noic acid, and 75 millimolar ole at pH 7.0. The amount of acrylamide bis acrylamide solution that we add to the buffer will depend on the percent gel that we are making to make the gradient gel.
We will make one 4%mix and one 13%mix, and the stacking gel will be a 3.5%mix. Do not forget to add glycerol to the 13%mix. When all three mixtures are ready, we add an appropriate amount of ammonium per sulfate and temid to the mixtures and pour the mixtures into a gradient former casting chamber.
The left part of the chamber contains the 5%mix. The right part of the chamber contains the 13%mix in a small magnetic stirring bar. We start to pour the gel by opening the right part of the chamber.
Then immediately we open the left part of the chamber and let the 5%solution mix with a 13%solution to make the gradient. We add a small amount of isopropanol to the top of the gel to form a sharp straight border for the gel after polymerization. Approximately one hour later when the polymerization of the gradient gel is complete, we remove the isopropanol and pour the 3.5 stacking gel into the apparatus and insert a gel comb.
We will assemble and run the gels in a cold room of about four to seven degrees Celsius. To run this gel, we'll be using two different buffers. In the anode chamber, we'll use a buffer containing 25 millimolar ole hydrochloric acid pH 7.0.
The cathode buffer will contain 50 millimeter tric, 7.5 millimolar ole hydrochloric acid, 0.02%weight per volume, doil beta D multiside or DDM and 0.05%weight to volume, deoxy coate pH 7.0. In order to prepare the supernatant to be run in the gel that we've just poured, loading buffer is added to yield a final concentration of 5%weight to volume glycerol and 0.01%weight to volume. Ponto S, we typically add a 10 x stock solution containing glycerol and poncho s directly to the supernatants.
Now we're ready to load the gel sample onto the gel. We will load the gel wells with a volume that contains 20 to 40 milligrams of the crude mitochondrial protein per lane. Immediately after loading, the gel is run at 100 volts.
Once the sample has entered the stacking gel, the voltage can be raised to 500 volts with the current limited to 15 milliamps. Although we usually run the gel overnight at 70 to 100 volts, we stop the electrophoresis when a sharp line of the red zos dye approaches the gel front. Now that the gel has been run, we can analyze the activity of different protein complexes directly in the gel.
The time of incubation for each assay depends on the efficiency of protein solubilization and the amount of material loaded per well. Complexes one, four and five usually yield stronger signals than complexes two and three. This is why staining time for complex two and three should be longer.
To test the complex activity, we will need to use specific buffers for each complex. You can find the ingredients for the individual buffers in the written protocol. Now we will cut out individual lanes of the gel To perform the Ingel activity assays separately.
For all the complexes, incubate the gel slice in their respective buffers for several hours at room temperature for all complexes except for complex five. After incubation, fix the gel in 50%methanol and 10%acetic acid for 15 to 45 minutes. The fixed gel is preserved in 10%acetic acid.
You see here how the staining of complex one appears. There is one deep purple band in the gel slice that corresponds to complex one. Clear gels let you perform densitometry from mitochondrial complex activities and make comparative analyses.
After dense cytometry, we usually dry the gel slices for long-term storage and or for scanning in order to obtain a high quality image complex, two stains the same purple color as one complex three and complex four. Both give an orange or yellowish staining band. Here are examples of complex two, three and four staining activities.
We have just shown you how to analyze the activity of mitochondrial exudative ation complexes, which were extracted from human embryonic stem cells and separated by higher resolution, including native gel electros. So that's it. Thank you for watching and good luck with your experiment.
