CELL CULTURE

We need to understand the principle of the cell culture. Cell culture is that we take cells outside the body, put them in nutrient medium and allow them to grow in the incubator outside the body. There is excuses for this type of method is that everyone can use certain study on the cell. It will be much safer if we can do the study/experiment outside, in virtual. All this procedures are not safe inside the body, so we take the cells and grow them outside the body. That is one aspect. The other aspect of cell culture, you get an infection and you go to the neurologist. He take the sample and grow it. He put it in the nutrient medium and then observe and identify what type of pathogen is present because its going to grow. So this is the basic principle and he saw what happened to them is that in virtual, the cell behaves in a similar function as they live in the body. *In virtual : outside the body *In vivo: inside the body The 1st line of cell which we take them from the body and grow them we called primary culture. This 1o culture, the cell we put them in nutrient medium and theyre going to divide and grow. After they grow, we put them in those small plate. They fill up the plate and we put an enzyme that liberated from the medium, then we transfer some of them to a new plate. The cells that we transfer to the new plate called 2o culture. Those cells behaves as if they were inside the body.

The doctor shows pictures of fibroblasts (slide 14)

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For example the fibroblasts which are the mother cells that give us heat, a connective tissue, in culture they begin to secrete collagen, their main protein that is present in connective tissue. Skeletal muscle theyre going to secrete contractile protein such as myosin, actin and etc.

The doctor shows pictures of myoblasts (slide 26) From the suffix myo means muscle. We said myoblasts. What we understand by the word blasts? It is an immature cell, undifferentiated, has the ability to divide and give us new cells of myocytes. Im sure you all know the hypertrophy that occur in cardiac muscle due to overload, in pathology. So this is myoblasts, how they occur in spindle shape , they start to secret protein, before that theyre multinucleated and the cell diffuse together and then we have embracynthium oligodendrocyte which are suppose to present in CNS etc.

Slide 27 The tobacco cell put in culture and we use fluorescent stain to stain their product, we see that its incorporated in chromatin. This just to show you, cell in culture behave as they were inside the body and they will secret their original contents.

FRACTIONATION We come to fractionation. It means we break the things into different component. We have study that the cell has cell membrane, nucleus, ribosomes, mitochondria. So in this in method, we extract only the desired component such as only mitochondria or only ribosomes etc. This is just to focus on a particular component of cells. So we break the compound. How we do that? In the beginning we subjected to osmotic shock or we can subjected to ultrasonic vibration and then break this component of the cell and then we put in a centrifuge and then we spin them. After we spin them at a certain speed. We begin at 1000g , the component will break. Some of them will settle down, the other will suspended in the fluid. Then, at this gradually state we take whatever suspended, put in a new centrifuge and spin it at a higher speed. Briefly, homogenization by breaking down the cell or by subjecting it to vibration and centrifuge. We spin them at 1000g only nuclei will precipitate. If we are going to study nuclei, just take the supernatant and spin them at 10000g mitochondria + lysosome will ppt. If we take supernatant over here and put at 100 000g microsomes: ER, ribosomes will ppt/separate.
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Slide 31 These are different steps where we start we put in test tube, we grind them, centrifuge it. *Slide 32* By this method of fractionation we can isolate different component of the cell. Now, the molecular of functional aspect , I said before functional component in the cell is a protein and the protein perform a function. In case I want to evaluate a certain a function for example in genetic, they have different steps in the formation of protein. From their transcription formation of mRNA go to the ribosome and then translation result in protein could be subjected to further modification example modified in Golgi body etc. There could be other factor that influenced the protein and could form false translation in protein modification. If something happen in this protein ; produced in lesser amount or false form, it will reflected on the function.

ANTIBODIES Antibody are commercially prepared, we can buy antibody for any protein. These antibodies is raised to recognize specific component in the cell. For example I want antibody to recognize myosin and actin etc. What do I do? This is called 1o antibody would specifically combine to the component it is raised against antigen antibody reaction. Now, I know the applied antibody combine to specific component in the cell. Where is it gone and I observe. I use another antibody that is called 2o antibody recognize the 1o antibody. For 2o antibody, I can put on it a tag make me see where it is gone. This tag could be a colour, could be fluorescent weve talked about fluorescent microscopy. The fluorescent ink that give me a colour when I look at it will specifically tell me where is the component of the cell associated either with ER or mitochondria or plasma membrane etc. I can look at it. So this is one aspect where I apply antigen-antibody reaction to locate the certain component of the cell whether it is present or not present and the intensity of that colour of what I see will reflect on how much of that component present. The other aspect is as we fractionate the cell, we break it down, what do we do? We extract only the protein and we put it in an electrical cell. The protein when theyre denatured, they unfolded and become a straight chain. Then if we subjected to electrical field, they will migrate from the +ve to the ve pole. Supposedly, I take 2 different person with weight difference. I asked them to run a same distance. Each will run according to their weight. Lighter weight will run faster. Same happen to protein. At beginning, we use antibody to recognize

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specific component of the cell. Then we use 2o antibody to recognize the 1o antibody and will have a tag for me to see it. This tag could be fluorescent/another dye etc.

As I said, protein are extracted and put in a electrical circuit, they have net +ve and ve charge and of course they break down and they back to their essential structure that is straight chain of amino acid. And the protein they are put in a field where they will migrate according to their molecular weight.

Slide 38 This is protein before theyre break down. Then we break down disulfide etc and will be straight chain. Here we have a box with polyacrylamide. This will connected to electrical source. Slide 41 Here we have several opening 1,2,3,4 etc. What you see here is the protein that is migrated in this gel. This is 1 protein, another protein, another protein. Summary, we take tissue. We homogenized it to break down the component. Then we extract the protein. The protein which I have extracted I take it to electrophores. I have protein for control, for experimental etc. Because I want to compare. Its the same protein but I want to see what happen to the protein under this condition and another condition. The 1 st thing over here, I used a molecular marker. They give different protein and this protein gives us molecular weight. In the 2nd thing, I put the control example. 3rd and 4th thing, I put the experimental. All this protein I put over here and then start the experiment by conducting the electricity of this box and then the protein which are probably denatured, theyll migrate each one according to its molecular weight. Eventually I use 2o antibody, as I said 1o antibody then the 2o , transfer to a membrane and then the end of reaction gives me black colour. This is the dye. The principle and step used, as I said homogenization extract the protein. Then I used selectively antibody I want, the 1o antibody will bind specifically to the component of the cell. Then I used 2o antibody that binds to the 1o antibody and 2o antibody has dye which enable mee to see it. This reaction could be a colour if I use microscopy it could be the black colour dye. Which I use the electrophores. Thats how I can evaluate a certain function.
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